Cd33 targeted immunotherapies

ABSTRACT

The present disclosure provides improved CD33 targeting polypeptides and compositions for adoptive T cell therapies for treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/898,392, filed Sep. 10, 2019, and U.S.Provisional Application No. 62/845,304, filed May 8, 2019, each of whichis incorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BLBD_119_02WO_ST25.txt. The text file is 302 KB,was created on May 5, 2020, and is being submitted electronically viaEFS-Web, concurrent with the filing of the specification.

BACKGROUND Technical Field

The present disclosure relates to improved adoptive cell therapiesdirected against CD33. More particularly, the disclosure relates toanti-CD33 VHH-containing chemically regulated signaling molecules,anti-CD33 VHH-containing chimeric antigen receptors, cells, and relatedmethods of treatment using the same.

Description of the Related Art

The global burden of cancer doubled between 1975 and 2000. Cancer is thesecond leading cause of morbidity and mortality worldwide, withapproximately 14.1 million new cases and 8.2 million cancer relateddeaths in 2012. The most common cancers are breast cancer, lung andbronchus cancer, prostate cancer, colon and rectum cancer, bladdercancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer,kidney and renal pelvis cancer, endometrial cancer, leukemia, andpancreatic cancer. The number of new cancer cases is projected to riseto 22 million within the next two decades.

Adoptive cellular therapy is emerging as a powerful paradigm fordelivering complex biological signals to treat cancer. In contrast tosmall molecule and biologic drug compositions, adoptive cell therapieshave the potential to execute unique therapeutic tasks owing to theirmyriad sensory and response programs and increasingly defined mechanismsof genetic control. Existing methods have focused primarily onscFv-based chimeric antigen receptors (CARs). CAR T cell therapy has metwith limited success due to poor CAR expression, in vivo expansion ofCART cells, rapid disappearance of the cells after infusion,disappointing clinical activity, and antigen escape.

There is a need to retrofit immune effector cells with improved CARarchitectures (CARchitectures) and/or improved machinery for sensing andintegrating chemical and/or biological information associated with localphysiological environments.

BRIEF SUMMARY

The present disclosure generally relates, in part, to VHH-baseddimerizing agent regulated immunoreceptor complexes (DARICs) andVHH-based chimeric antigen receptors (CARs) directed against CD33,polynucleotides encoding the same, compositions thereof, and methods ofmaking and using the same to treat cancer.

In particular embodiments, a VHH DARIC or VHH CAR binds full-lengthCD33. In particular embodiments, a VHH DARIC or VHH CAR binds a CD33splice variant. In certain embodiments, the CD33 splice variant lacksthe 124 amino acids encoded by exon 2 of the human CD33 gene (CD33 C2variant). In certain embodiments, the CD33 splice variant lacks 54carboxy-terminal amino acids due to an early translation stop signalresiding in exon 7a. In certain embodiments, the CD33 splice variantlacks the 124 amino acids encoded by exon 2 and 54 carboxy-terminalamino acids due to an early translation stop signal residing in exon 7a.

In particular embodiments, a VHH DARIC or VHH CAR binds both full-lengthCD33 and a CD33 splice variant.

In various embodiments, a non-natural cell comprises: a firstpolypeptide comprising: an FRB multimerization domain polypeptide orvariant thereof; a CD8a transmembrane domain or a CD4 transmembranedomain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signalingdomain; and a second polypeptide comprising: an anti-CD33 VHH antibodythat has an amino acid sequence set forth in any one of SEQ ID NOs:2-21; an FKBP multimerization domain polypeptide or variant thereof; anda CD4 transmembrane domain or a CD8a transmembrane domain; wherein abridging factor promotes the formation of a polypeptide complex on thenon-natural cell surface with the bridging factor associated with anddisposed between the multimerization domains of the first and secondpolypeptides.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 20.

In particular embodiments, the FKBP multimerization domain is FKBP12.

In some embodiments, the FRB polypeptide is FRB T2098L.

In certain embodiments, the bridging factor is selected from the groupconsisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

In various embodiments, the first polypeptide comprises a signalpeptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; anda CD3ζ primary signaling domain.

In particular embodiments, the second polypeptide comprises a signalpeptide and a CD4 transmembrane domain.

In further embodiments, the second polypeptide comprises a costimulatorydomain.

In some embodiments, the costimulatory domain of the second polypeptideis selected from a costimulatory molecule selected from the groupconsisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94,CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10(DAP10), Linker for activation of T-cells family member 1 (LAT), SH2Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptorassociated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18,TNRFS25, and zeta chain of T cell receptor associated protein kinase 70(ZAP70).

In additional embodiments, the costimulatory domain of the secondpolypeptide is a costimulatory domain isolated from OX40 or TNFR2.

In further embodiments, the second polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 22-31.

In particular embodiments, the second polypeptide comprises the sequenceset forth in SEQ ID NO: 30.

In preferred embodiments, a first polypeptide comprises an amino acidsequence set forth in SEQ ID NO 82.

In various embodiments, a non-natural cell comprises a polypeptidecomplex that comprises: a first polypeptide comprising: an FRBmultimerization domain polypeptide or variant thereof; a CD8atransmembrane domain or a CD4 transmembrane domain; a CD137co-stimulatory domain; and/or a CD3ζ primary signaling domain; a secondpolypeptide comprising: an anti-CD33 VHH antibody that has an amino acidsequence set forth in any one of SEQ ID NOs: 2-21; an FKBPmultimerization domain polypeptide or variant thereof; and a CD4transmembrane domain or a CD8a transmembrane domain; and a bridgingfactor associated with and disposed between the multimerization domainsof the first and second polypeptides.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 20.

In particular embodiments, the FKBP multimerization domain is FKBP12.

In certain embodiments, the FRB polypeptide is FRB T2098L.

In some embodiments, the bridging factor is selected from the groupconsisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

In additional embodiments, the first polypeptide comprises a signalpeptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; anda CD3ζ primary signaling domain.

In particular embodiments, the second polypeptide comprises a signalpeptide and a CD4 transmembrane domain.

In some embodiments, the second polypeptide comprises a costimulatorydomain.

In various embodiments, the costimulatory domain of the secondpolypeptide is selected from a costimulatory molecule selected from thegroup consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain familymember 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83,CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptorassociated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18,TNRFS25, and zeta chain of T cell receptor associated protein kinase 70(ZAP70).

In additional embodiments, the costimulatory domain of the secondpolypeptide is a costimulatory domain isolated from OX40 or TNFR2.

In further embodiments, the second polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 22-31.

In particular embodiments, the second polypeptide comprises the sequenceset forth in SEQ ID NO: 30.

In preferred embodiments, a first polypeptide comprises an amino acidsequence set forth in SEQ ID NO 82.

In certain embodiments, the cell is a hematopoietic cell.

In particular embodiments, the cell is a T cell, an αβ T cell, or a γδ Tcell.

In further embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.

In various embodiments, the cell is an immune effector cell.

In some embodiments, the cell is a cytotoxic T lymphocytes (CTLs), atumor infiltrating lymphocytes (TILs), or a helper T cell.

In additional embodiments, the cell is a natural killer (NK) cell ornatural killer T (NKT) cell.

In various embodiments, the source of the cell is peripheral bloodmononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymusissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, or tumors.

In particular embodiments, the FRB multimerization domain and the FKBPmultimerization domain localize extracellularly when of the firstpolypeptide and the second polypeptide are expressed.

In some embodiments, a fusion polypeptide comprises: a first polypeptidecomprising: an FRB multimerization domain polypeptide or variantthereof; a CD8a transmembrane domain or a CD4 transmembrane domain; aCD137 co-stimulatory domain; and/or a CD3ζ primary signaling domain; apolypeptide cleavage signal; and a second polypeptide comprising: ananti-CD33 VHH antibody that has an amino acid sequence set forth in anyone of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide orvariant thereof; and a CD4 transmembrane domain or a CD8a transmembranedomain.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the FKBP multimerization domain is FKBP12.

In certain embodiments, the FRB polypeptide is FRB T2098L.

In some embodiments, the bridging factor is selected from the groupconsisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

In additional embodiments, the first polypeptide comprises a signalpeptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; anda CD3ζ primary signaling domain.

In particular embodiments, the second polypeptide comprises a signalpeptide and a CD4 transmembrane domain.

In certain embodiments, the fusion polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 32-41.

In certain embodiments, the fusion polypeptide comprises the sequenceset forth in SEQ ID NO: 40.

In further embodiments, the second polypeptide comprises a costimulatorydomain.

In various embodiments, the costimulatory domain of the secondpolypeptide is selected from a costimulatory molecule selected from thegroup consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain familymember 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83,CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptorassociated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18,TNRF S25, and zeta chain of T cell receptor associated protein kinase 70(ZAP70).

In additional embodiments, the costimulatory domain of the secondpolypeptide is a costimulatory domain isolated from OX40 or TNFR2.

In certain embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving 2A polypeptide.

In various embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide selected from the group consisting of: afoot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis Avirus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, aporcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide,and an encephalomyocarditis virus 2A peptide.

In some embodiments, the fusion polypeptide comprises the sequence setforth in any one of SEQ ID NOs: 42-61.

In some embodiments, the fusion polypeptide comprises the sequence setforth in any one of SEQ ID NOs: 50 or 60.

In further embodiments, the FRB multimerization domain and the FKBPmultimerization domain localize extracellularly when of the firstpolypeptide and the second polypeptide are expressed.

In various embodiments, polypeptide complex comprises: a firstpolypeptide comprising: an FRB multimerization domain polypeptide orvariant thereof; a CD8a transmembrane domain or a CD4 transmembranedomain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signalingdomain; a second polypeptide comprising: an anti-CD33 VHH antibody thathas an amino acid sequence set forth in any one of SEQ ID NOs: 2-21;

an FKBP multimerization domain polypeptide or variant thereof; and a CD4transmembrane domain or a CD8a transmembrane domain; and a bridgingfactor associated with and disposed between the multimerization domainsof the first and second polypeptides.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the FKBP multimerization domain is FKBP12.

In additional embodiments, the FRB polypeptide is FRB T2098L.

In particular embodiments, the bridging factor is selected from thegroup consisting of: AP21967, sirolimus, everolimus, novolimus,pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, andzotarolimus.

In certain embodiments, the first polypeptide comprises a CD8atransmembrane domain; a CD137 co-stimulatory domain; and a CD3ζ primarysignaling domain.

In various embodiments, the second polypeptide comprises a CD4transmembrane domain.

In further embodiments, the second polypeptide comprises a costimulatorydomain.

In some embodiments, the costimulatory domain of the second polypeptideis selected from a costimulatory molecule selected from the groupconsisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94,CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10(DAP10), Linker for activation of T-cells family member 1 (LAT), SH2Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptorassociated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18,TNRFS25, and zeta chain of T cell receptor associated protein kinase 70(ZAP70).

In particular embodiments, the costimulatory domain of the secondpolypeptide is a costimulatory domain isolated from OX40 or TNFR2.

In certain embodiments, the cell is a hematopoietic cell.

In various embodiments, the cell is a T cell, an αβ T cell, or a γδ Tcell.

In various embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.

In additional embodiments, the cell is an immune effector cell.

In some embodiments, the cell is a cytotoxic T lymphocytes (CTLs), atumor infiltrating lymphocytes (TILs), or a helper T cell.

In particular embodiments, the cell is a natural killer (NK) cell ornatural killer T (NKT) cell.

In additional embodiments, the source of the cell is peripheral bloodmononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymusissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, or tumors.

In further embodiments, the FRB multimerization domain and the FKBPmultimerization domain localize extracellularly when of the firstpolypeptide and the second polypeptide are expressed.

In preferred embodiments, a first polypeptide comprises an amino acidsequence set forth in SEQ ID NO 82.

In particular embodiments, a chimeric antigen receptor (CAR) comprises:an anti-CD33 VHH antibody that has an amino acid sequence set forth inany one of SEQ ID NOs: 2-21; a hinge domain; a transmembrane domain; oneor more intracellular costimulatory signaling domains; and/or a primarysignaling domain.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 20.

In various embodiments, the CAR comprises from 5′ to 3′: an anti-CD33VHH antibody that has an amino acid sequence set forth in any one of SEQID NOs: 2-21; a hinge domain; a transmembrane domain; one or moreintracellular costimulatory signaling domains; and/or a primarysignaling domain.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 10.

In particular embodiments, the anti-CD33 VHH antibody has an amino acidsequence set forth in SEQ ID NO: 20.

In certain embodiments, the hinge domain and transmembrane domain areisolated from CD8a, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80,CD86, CD 134, CD137, CD152, CD154, AMN, and PD1.

In additional embodiments, the one or more costimulatory signalingdomains are isolated from a costimulatory molecule selected from thegroup consisting of: CD28, CD134, CD137, and CD278.

In particular embodiments, the CAR comprises a CD8a signal peptide, aCD8a hinge and transmembrane domain, a CD134 costimulatory domain, and aCD3ζ primary signaling domain.

In further embodiments, a CAR comprises the amino acid sequence setforth in any one of SEQ ID NOs: 62-81.

In further embodiments, a CAR comprises the amino acid sequence setforth in any one of SEQ ID NOs: 70 or 80.

In some embodiments, polynucleotide encoding a first or secondpolypeptide, a fusion polypeptide, or a CAR contemplated herein isprovided.

In various embodiments, a cDNA encoding a first or second polypeptide, afusion polypeptide, or a CAR contemplated herein is provided.

In particular embodiments, an RNA encoding a first or secondpolypeptide, a fusion polypeptide, or a CAR contemplated herein isprovided.

In additional embodiments, a vector comprising a polynucleotidecontemplated herein is provided.

In certain embodiments, the vector is an expression vector.

In certain embodiments, the vector is a transposon.

In further embodiments, the vector is a piggyBAC transposon or aSleeping Beauty transposon.

In particular embodiments, the vector is a viral vector.

In particular embodiments, the vector is an adenoviral vector, anadeno-associated viral (AAV) vector, a herpes virus vector, a vacciniavirus vector, or a retroviral vector.

In additional embodiments, the retroviral vector is a lentiviral vector.

In various embodiments, the lentiviral vector is selected from the groupconsisting of: human immunodeficiency virus 1 (HIV-1); humanimmunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (Hy); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV).

In further embodiments, a cell comprising a first and secondpolypeptide, a fusion polypeptide, or a CAR contemplated herein isprovided.

In particular embodiments, the cell is a hematopoietic cell.

In certain embodiments, the cell is an immune effector cell.

In various embodiments, the cell is a T cell, an αβ T cell, or a γδ Tcell.

In some embodiments, the cell expresses CD3+, CD4+, CD8+, or acombination thereof.

In particular embodiments, the cell is a cytotoxic T lymphocyte (CTL), atumor infiltrating lymphocyte (TIL), or a helper T cell.

In further embodiments, the cell is a natural killer (NK) cell ornatural killer T (NKT) cell.

In certain embodiments, a composition comprises a cell contemplatedherein.

In particular embodiments, a composition comprises a physiologicallyacceptable carrier and a cell contemplated herein.

In additional embodiments, method of treating a subject in need thereofcomprising administering the subject an effective amount of acomposition contemplated herein.

In particular embodiments, a method of treating, preventing, orameliorating at least one symptom of a cancer, infectious disease,autoimmune disease, inflammatory disease, and immunodeficiency, orcondition associated therewith, comprises administering to the subjectan effective amount of a composition contemplated herein.

In some embodiments, a method of treating a solid cancer comprisesadministering to the subject an effective amount of a compositioncontemplated herein.

In various embodiments, the solid cancer is selected from the groupconsisting of: lung cancer, liver cancer, gastric cancer, colorectalcancer, head and neck cancer, urothelial cancer, prostate cancer,testicular cancer, endometrial cancer, pancreatic cancer, breast cancer,cervical cancer, ovarian cancer, skin cancer, and melanoma.

In certain embodiments, a method of treating a hematological malignancycomprises administering to the subject an effective amount of acomposition contemplated herein.

In various embodiments, the hematological malignancy is a leukemia,lymphoma, or multiple myeloma.

In particular embodiments, the hematological malignancy is acutemyelogenous leukemia (AML).

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows a cartoon of a VHH-DARIC polypeptide complex.

FIG. 1B shows a cartoon of a CD33 VHH DARIC architecture.

FIG. 2A shows the expression of CD33 VHH1-5 DARICs in transduced Tcells, as detected by anti-VHH staining (top row) and by CD33-Fc binding(bottom row).

FIG. 2B shows the expression of CD33 VHH9-10 DARICs in transduced Tcells, as detected by CD33-Fc binding.

FIG. 3A shows the phenotype of T cells transduced with CD33 VHH1-5DARICs or controls.

FIG. 3B shows the phenotype of T cells transduced with CD33 VHH9-10DARICs or controls.

FIG. 4A shows IFNγ secretion from CD33 VHH1-5 DARICs or control cellscultured with CD33⁺ THP-1 cells at an E:T ratio of 1:1 in the presenceor absence of AP21967 for 24 hours.

FIG. 4B shows IFNγ secretion from CD33 VHH9-10 DARICs or control cellscultured with CD33⁺ THP-1 cells at an E:T ratio of 1:1 in the presenceor absence of AP21967 for 24 hours.

FIG. 4C shows IFNγ secretion from CD33 VHH9-10 DARICs or control cellscultured with modified 293T cells that express full-length CD33 (CD33M)or a CD33 splice variant (CD33m, C2) at an E:T ratio of 1:1 in thepresence or absence of AP21967 for 24 hours.

FIG. 5A shows CD33 expression on MV4-11 cells, MV4-11 cells engineeredto knock out the CD33 gene (CD33-KO cells), and in an unstained control.

FIG. 5B shows IFNγ secretion from anti-CD33 VHH9 DARIC T cells or UTD Tcells co-cultured with MV4-11 cells or CD33-KO cells at an E:T ratio of1:1 in the presence or absence of AP21967 for 24 hours.

FIG. 5C shows IFNγ secretion from UTD T cells, anti-CD33 CAR T cells, oranti-CD33 VHH DARIC T cells co-cultured with MV4-11 cells (left panel)or CD33-KO cells engineered to express a CD33m splice variant(CD33-KO-C2 cells; right panel) at an E:T ratio of 1:1 in the presenceor absence of AP21967 for 24 hours.

FIG. 6 shows IFNγ secretion from anti-CD33 VHH DARIC T cells co-culturedwith CD33⁺ THP-1 cells at an E:T ratio of 1:1 in the presence or absenceof soluble CD33 (CD33-Fc) and AP21967 for 24 hours.

FIG. 7 shows IFNγ secretion from anti-CD33 VHH DARIC T cells co-culturedwith CD33^(neg) 293T cells transfected with different amounts of mRNAencoding CD33 at an E:T ratio of 1:1 in the presence or absence ofAP21967 for 24 hours.

FIG. 8A shows tumor growth measured as a function of luminescence inimmunodeficient NSG mice inoculated with HL60 AML, tumor cellsexpressing a luciferase reporter and treated, 10 days post-inoculation(Day 0), with UTD T cells or anti-CD33 VHH DARIC T cells in the absenceof rapamycin.

FIG. 8B shows tumor growth measured as a function of luminescence inimmunodeficient NSG mice inoculated with HL60 AML tumor cells expressinga luciferase reporter and treated, 10 days post-inoculation (Day 0),with UTD T cells or anti-CD33 VHH DARIC T cells and 0.1 mg/kg rapamycin.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for full-length humanCD33.

SEQ ID NOs: 2-21 set forth the amino acid sequences for an anti-CD33 VHHdomains.

SEQ ID NOs: 22-31 set forth the amino acid sequences for anti-CD33 VHHDARIC binding components.

SEQ ID NOs: 32-41 set forth the amino acid sequences for anti-CD33 VHHDARIC fusion proteins.

SEQ ID NOs: 42-51 set forth the amino acid sequences for anti-CD33 VHHDARIC.OX40 fusion proteins.

SEQ ID NOs: 52-61 set forth the amino acid sequences for anti-CD33 VHHDARIC.TNFR2 fusion proteins.

SEQ ID NOs: 62-81 set forth the amino acid sequences for anti-CD33 VHHCARS.

SEQ ID NO: 82 sets forth the amino acid sequence for an anti-CD33 VHHDARIC signaling component.

SEQ ID NO: 83 sets forth the polynucleotide sequence for a Kozaksequence.

SEQ ID NOs: 84-94 set forth the amino acid sequences of various linkers.

SEQ ID NOs: 95-119 set forth the amino acid sequences of proteasecleavage sites and self-cleaving polypeptide cleavage sites.

In the foregoing sequences, Xaa, if present, may refer to any amino acidor the absence of an amino acid. In preferred embodiments, XaaXaa refersto the amino acid sequence SS or KP.

DETAILED DESCRIPTION A. Overview

Cancer is among the leading causes of death worldwide. About 10% ofcancers are hematological malignancies, which includes leukemia,lymphomas, and myelomas. Acute myeloid leukemia (AML) is the most commonand fatal hematological malignancy in adults. Despite major scientificdiscoveries and novel therapies over the past four decades, thetreatment outcomes of AML, especially in the adult patient populationremain dismal. Standard chemotherapies can induce complete remission inselected patients; however, a majority of patients eventually relapseand succumb to the disease. In 2012, the worldwide incidence of AML wasabout 351,965 and about 265,461 people died from AML.

CD33 is expressed on the majority of acute myeloid leukemia (AML)leukemic blasts and, possibly, leukemic stem cells. CD33 is achallenging target because of its low expression and slowinternalization; these characteristics limit antibody-dependentcell-mediated cytotoxicity and intracellular drug accumulation and,consequently, the activity of unlabeled and toxin-carrying antibodies.

The disclosure generally relates to improved compositions and methodsfor regulating the spatial and temporal control of adoptive celltherapies using dimerizing agent regulated immunoreceptor complexes(DARICs) that bind CD33. Without wishing to be bound by any particulartheory, DARIC compositions and methods contemplated herein providenumerous advantages over CAR T cell therapies existing in the art,including but not limited to, both spatial and temporal control overimmune effector cell signal transduction binding and signalingactivities. DARIC temporal control primes the DARIC machinery forsignaling through bridging factor mediated association of a DARICbinding component to a DARIC signaling component. DARIC spatial controlengages the signaling machinery through recognition of CD33 by the DARICbinding domain of the DARIC binding component. In this manner, DARICimmune effector cells become activated when both a target cellexpressing CD33 and a bridging factor are present.

The disclosure also relates to improved anti-CD33 CAR architectures thatovercome potential limitations of existing CAR T therapies including butnot limited to tonic signaling or antigen independent signaling, poorexpression and/or subtherapeutic activity.

In various embodiments, the disclosure contemplates anti-CD33 VHH DARICsor anti-CD33 VHH CARS that generate an anti-cancer response againstcancers, e.g., AML that express CD33, e.g., full-length CD33 and/or aCD33 splice variant.

In particular embodiments, a DARIC includes a polypeptide (DARICsignaling component) that comprises a multimerization domain polypeptideor variant thereof, a transmembrane domain, a costimulatory domain;and/or a primary signaling domain; and a polypeptide (DARIC bindingcomponent) that comprises an anti-CD33 VHH, a multimerization domainpolypeptide or variant thereof, a transmembrane domain; and optionally acostimulatory domain. In the presence of a bridging factor, the DARICbinding and signaling components associate with one another through thebridging factor to form a functionally active DARIC that targets cellsexpressing CD33.

In particular embodiments, the multimerization domains of the DARICbinding and DARIC signaling components are positioned extracellularly.Extracellular position of the multimerization domains provides numerousadvantages over intracellular positioning including, but not limited to,more efficient positioning of the anti-CD33 VHH domain, higher temporalsensitivity to bridging factor regulation, and less toxicity due toability to use non-immunosuppressive doses of particular bridgingfactors.

Polynucleotides encoding DARICs, DARIC binding components, and DARICsignaling components; DARIC binding components, DARIC signalingcomponents, DARIC protein complexes, DARIC fusion proteins; cellscomprising polynucleotides encoding DARICs, DARIC binding components,and DARIC signaling components and/or expressing the same; and methodsof using the same to treat an immune disorder are contemplated herein.

Techniques for recombinant (i.e., engineered) DNA, peptide andoligonucleotide synthesis, immunoassays, tissue culture, transformation(e.g., electroporation, lipofection), enzymatic reactions, purificationand related techniques and procedures may be generally performed asdescribed in various general and more specific references inmicrobiology, molecular biology, biochemistry, molecular genetics, cellbiology, virology and immunology as cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wileyand Sons, updated July 2008); Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: APractical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA,1985); Current Protocols in Immunology (Edited by: John E. Coligan, AdaM. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology andApplications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders,2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for theAnalysis of Complex Genomes, (Academic Press, New York, 1992); Guthrieand Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press,New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); NucleicAcid The Hybridization (B. Hames & S. Higgins, Eds., 1985);Transcription and Translation (B. Hames & S. Higgins, Eds., 1984);Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz,2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park,Ed., 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRLPress, 1986); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane,Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayerand Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C C Blackwell,eds., 1986); Roitt, Essential Immunology, 6th Edition, (BlackwellScientific Publications, Oxford, 1988); Current Protocols in Immunology(Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W.Strober, eds., 1991); Annual Review of Immunology; as well as monographsin journals such as Advances in Immunology.

B. Definitions

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of particular embodiments, preferred embodimentsof compositions, methods and materials are described herein. For thepurposes of the present disclosure, the following terms are definedbelow.

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one, or to one or more) of thegrammatical object of the article. By way of example, “an element” meansone element or one or more elements.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “and/or” should be understood to mean either one, or both ofthe alternatives.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

In one embodiment, a range, e.g., 1 to 5, about 1 to 5, or about 1 toabout 5, refers to each numerical value encompassed by the range. Forexample, in one non-limiting and merely illustrative embodiment, therange “1 to 5” is equivalent to the expression 1, 2, 3, 4, 5; or 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

As used herein, the term “substantially” refers to a quantity, level,value, number, frequency, percentage, dimension, size, amount, weight orlength that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher compared to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, “substantially the same” refers to a quantity, level, value,number, frequency, percentage, dimension, size, amount, weight or lengththat produces an effect, e.g., a physiological effect, that isapproximately the same as a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are present that materially affect the activity or action ofthe listed elements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. It is also understoodthat the positive recitation of a feature in one embodiment, serves as abasis for excluding the feature in a particular embodiment.

An “antigen (Ag)” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T cell response in ananimal, including compositions (such as one that includes acancer-specific protein) that are injected or absorbed into an animal.Exemplary antigens include but are not limited to lipids, carbohydrates,polysaccharides, glycoproteins, peptides, or nucleic acids. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedantigens.

A “target antigen” or “target antigen of interest” refers to a portionof CD33, that a binding domain contemplated herein, is designed to bind.In particular embodiments, the target antigen is an epitope of the aminoacid sequence set forth in SEQ ID NO: 1.

“CD33” refers to a cell surface receptor also known assialic-acid-binding immunoglobulin-like lectin 3 (SIGLEC-3) or GP67. TheCD33 gene is located on chromosome 19 and produces a glycosylatedprotein of about 67 kD. CD33 has two Ig-like domains, one V-set domainand one C2-set domain. CD33 plays a role in mediating cell-cellinteractions and in maintaining immune cells in a resting state. CD33recognizes and binds alpha-2,3- and more avidly alpha-2,6-linked sialicacid-bearing glycans. Upon engagement of ligands such as Clq orsialylated glycoproteins, two immunoreceptor tyrosine-based inhibitorymotifs (ITIMs) located in CD33 cytoplasmic tail are phosphorylated bySrc-like kinases such as LCK. These phosphorylations provide dockingsites for the recruitment and activation of protein-tyrosinephosphatases PTPN6/SHP-1 and PTPN11/SHP-2. CD33 also has at least threeidentified splice variants. CD33^(ΔE2) splice variant lacks the aminoacid sequence encoded by exon 2 of the human CD33 gene (amino acids13-139 of full-length CD33; e.g., NP 001076087.1, C2). CD33⁷a splicevariant lacks 54 carboxy-terminal amino acids due to an earlytranslation stop signal residing in exon 7a (e.g., NP_001171079.1).CD33^(ΔE2/7a) lacks the amino acids encoded by exon 2 and 54carboxy-terminal amino acids. CD33 is normally expressed on subsets ofnormal B cells and activated T cells and natural killer cells but is notexpressed on hematopoietic stem cells or outside the hematopoieticsystem. Both full-length CD33 and/or CD33 splice variants are alsoexpressed in acute myeloid leukemia (AML) blast cells in a majority ofAML patients.

An “antibody” refers to a binding agent that is a polypeptide comprisingat least a light chain or heavy chain immunoglobulin variable regionwhich specifically recognizes and binds an epitope of an antigen, suchas a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, ornucleic acid containing an antigenic determinant, such as thoserecognized by an immune cell.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain or antigen binding fragments thereof.

A “heavy chain antibody” refers to an antibody that contains two V_(H)domains and no light chains (Riechmann L. et al, J. Immunol. Methods231:25-38 (1999); WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079). A“camelid antibody” refers to an antibody isolated from a Camel, Alpaca,or Llama that contains two V_(H) domains and no light chains. A“humanized VHH” or “humanized camelid antibody” refers to a non-humanVHH or camelid antibody that has undergone humanization to reducepotential immunogenicity of the antibody in human recipients.

A “V_(H)H,” “V_(H)H antibody,” or “V_(H)H domain” as used herein refersan antibody fragment that contains the smallest known antigen-bindingunit of the variable region of a heavy chain antibody (Koch-Nolte, etal, FASEB J., 21: 3490-3498 (2007)).

A “linker” refers to a plurality of amino acid residues between thevarious polypeptide domains added for appropriate spacing andconformation of the molecule. In particular embodiments, a linkerseparates one or more VHH domains, hinge domains, multimerizationdomains, transmembrane domains, co-stimulatory domains, and/or primarysignaling domains.

Illustrated examples of linkers suitable for use in particularembodiments contemplated herein include, but are not limited to thefollowing amino acid sequences: GGG; DGGGS (SEQ ID NO: 84); TGEKP (SEQID NO: 85) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ IDNO: 86) (Pomerantz et al. 1995, supra); (GGGGS)_(n) wherein n=1, 2, 3, 4or 5 (SEQ ID NO: 87) (Kim et al., PNAS 93, 1156-1160 (1996.);EGKSSGSGSESKVD (SEQ ID NO: 88) (Chaudhary et al., 1990, Proc. Natl.Acad. Sci. U.S.A. 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 89)(Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 90);LRQRDGERP (SEQ ID NO: 91); LRQKDGGGSERP (SEQ ID NO: 92); LRQKD(GGGS)₂ERP (SEQ ID NO: 93). Alternatively, flexible linkers can be rationallydesigned using a computer program capable of modeling both DNA-bindingsites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260(1993), PNAS 91:11099-11103 (1994) or by phage display methods. In oneembodiment, the linker comprises the following amino acid sequence:GSTSGSGKPGSGEGSTKG (SEQ ID NO: 94) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

A “spacer domain,” refers to a polypeptide that separates two domains.In one embodiment, a spacer domain moves a VHH domain away from theeffector cell surface to enable proper cell/cell contact, antigenbinding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419).In particular embodiments, a spacer domain separates one or more VHHdomains, multimerization domains, transmembrane domains, co-stimulatorydomains, and/or primary signaling domains. The spacer domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource. In certain embodiments, a spacer domain is a portion of animmunoglobulin, including, but not limited to, one or more heavy chainconstant regions, e.g., CH2 and CH3. The spacer domain can include theamino acid sequence of a naturally occurring immunoglobulin hinge regionor an altered immunoglobulin hinge region.

A “hinge domain,” refers to a polypeptide that plays a role inpositioning the antigen binding domain away from the effector cellsurface to enable proper cell/cell contact, antigen binding andactivation. In particular embodiments, polypeptides may comprise one ormore hinge domains between the binding domain and the multimerizationdomain, between the binding domain and the transmembrane domain (TM), orbetween the multimerization domain and the transmembrane domain. Thehinge domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The hinge domain can include theamino acid sequence of a naturally occurring immunoglobulin hinge regionor an altered immunoglobulin hinge region.

A “multimerization domain,” as used herein, refers to a polypeptide thatpreferentially interacts or associates with another differentpolypeptide directly or via a bridging molecule, e.g., a chemicallyinducible dimerizer, wherein the interaction of differentmultimerization domains substantially contributes to or efficientlypromotes multimerization (i.e., the formation of a dimer, trimer, ormultipartite complex, which may be a homodimer, heterodimer, homotrimer,heterotrimer, homomultimer, heteromultimer). A multimerization domainmay be derived either from a natural, synthetic, semi-synthetic, orrecombinant source.

Illustrative examples of multimerization domains suitable for use inparticular embodiments contemplated herein include an FK506 bindingprotein (FKBP) polypeptide or variants thereof, an FKBP-rapamycinbinding (FRB) polypeptide or variants thereof, a calcineurin polypeptideor variants thereof, a cyclophilin polypeptide or variants thereof, abacterial dihydrofolate reductase (DHFR) polypeptide or variantsthereof, a PYR1-like 1 (PYL1) polypeptide or variants thereof, anabscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, aGIB1 polypeptide or variants thereof, or a GAI polypeptide or variantsthereof.

As used herein, the term “FKBP-rapamycin binding polypeptide” refers toan FRB polypeptide. In particular embodiments, the FRB polypeptide is anFKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for usein particular embodiments contemplated herein generally contain at leastabout 85 to about 100 amino acid residues. In certain embodiments, theFRB polypeptide comprises a 93 amino acid sequence Ile-2021 through Lys−2113 and a mutation of T2098L, with reference to GenBank Accession No.L34075.1. An FRB polypeptide contemplated herein binds to an FKBPpolypeptide through a bridging factor, thereby forming a ternarycomplex.

As used herein, the term “FK506 binding protein” refers to an FKBPpolypeptide. In particular embodiments, the FKBP polypeptide is anFKBP12 polypeptide or an FKBP12 polypeptide comprising an F36V mutation.In certain embodiments, an FKBP domain may also be referred to as a“rapamycin binding domain”. Information concerning the nucleotidesequences, cloning, and other aspects of various FKBP species is knownin the art (see, e.g., Staendart et al., Nature 346:671, 1990 (humanFKBP12); Kay, Biochem. J. 314:361, 1996). An FKBP polypeptidecontemplated herein binds to an FRB polypeptide through a bridgingfactor, thereby forming a ternary complex.

A “bridging factor” refers to a molecule that associates with and thatis disposed between two or more multimerization domains. In particularembodiments, multimerization domains substantially contribute to orefficiently promote formation of a polypeptide complex only in thepresence of a bridging factor. In particular embodiments,multimerization domains do not contribute to or do not efficientlypromote formation of a polypeptide complex in the absence of a bridgingfactor. Illustrative examples of bridging factors suitable for use inparticular embodiments contemplated herein include, but are not limitedto AP21967, rapamycin (sirolimus) or a rapalog thereof, coumermycin or aderivative thereof, gibberellin or a derivative thereof, abscisic acid(ABA) or a derivative thereof, methotrexate or a derivative thereof,cyclosporin A or a derivative thereof, FKCsA or a derivative thereof,trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivativethereof, or any combination thereof.

Rapamycin analogs (rapalogs) include, but are not limited to, thosedisclosed in U.S. Pat. No. 6,649,595, which rapalog structures areincorporated herein by reference in their entirety. In certainembodiments, a bridging factor is a rapalog with substantially reducedimmunosuppressive effect as compared to rapamycin. In a preferredembodiment, the rapalog is AP21967 (also known asC-16-(S)-7-methylindolerapamycin, IC₅₀=10 nM, a chemically modifiednon-immunosuppressive rapamycin analogue). Other illustrative rapalogssuitable for use in particular embodiments contemplated herein include,but are not limited to, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

A “substantially reduced immunosuppressive effect” refers to at leastless than 0.1 to 0.005 times the immunosuppressive effect observed orexpected for the same dose measured either clinically or in anappropriate in vitro (e.g., inhibition of T cell proliferation) or invivo surrogate of human immunosuppressive activity.

A “transmembrane domain” or “TM domain” is a domain that anchors apolypeptide to the plasma membrane of a cell. The TM domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource.

The term “effector function” or “effector cell function” refers to aspecialized function of an immune effector cell. Effector functionincludes, but is not limited to, activation, cytokine production,proliferation and cytotoxic activity, including the release of cytotoxicfactors, or other cellular responses elicited with antigen binding tothe receptor expressed on the immune effector cell.

An “intracellular signaling domain” or “endodomain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces an effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of anintracellular signaling domain necessary or sufficient to transduce aneffector function signal.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingdomains: primary signaling domains that initiate antigen-dependentprimary activation through the TCR (e.g., a TCR/CD3 complex) andco-stimulatory signaling domains that act in an antigen-independentmanner to provide a secondary or co-stimulatory signal.

A “primary signaling domain” refers to an intracellular signaling domainthat regulates the primary activation of the TCR complex either in astimulatory way, or in an inhibitory way. Primary signaling domains thatact in a stimulatory manner may contain signaling motifs which are knownas immunoreceptor tyrosine-based activation motifs or ITAMs.Illustrative examples of ITAM containing primary signaling domains thatare suitable for use in particular embodiments include, but are notlimited to those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, and CD66d.

As used herein, the term, “co-stimulatory signaling domain,” or“co-stimulatory domain” refers to an intracellular signaling domain of aco-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules from which co-stimulatory domains may beisolated include, but are not limited to: Toll-like receptor 1 (TLR1),TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspaserecruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28,CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278(ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation ofT-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte ProteinOf 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1(TRAT1), TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNFreceptor superfamily member 18 (TNFRS18; GITR), TNF receptor superfamilymember 25 (TNFRS25; DR3), and zeta chain of T cell receptor associatedprotein kinase 70 (ZAP70).

An “immune disorder” refers to a disease that evokes a response from theimmune system. In particular embodiments, the term “immune disorder”refers to a cancer, an autoimmune disease, or an immunodeficiency.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues.

As used herein, the term “malignant” refers to a cancer in which a groupof tumor cells display one or more of uncontrolled growth (i.e.,division beyond normal limits), invasion (i.e., intrusion on anddestruction of adjacent tissues), and metastasis (i.e., spread to otherlocations in the body via lymph or blood). As used herein, the term“metastasize” refers to the spread of cancer from one part of the bodyto another. A tumor formed by cells that have spread is called a“metastatic tumor” or a “metastasis.” The metastatic tumor containscells that are like those in the original (primary) tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” refers to an individual cell of a cancerous growth ortissue. Cancer cells include both solid cancers and liquid cancers. A“tumor” or “tumor cell” refers generally to a swelling or lesion formedby an abnormal growth of cells, which may be benign, pre-malignant, ormalignant. Most cancers form tumors, but liquid cancers, e.g., leukemia,do not necessarily form tumors. For those cancers that form tumors, theterms cancer (cell) and tumor (cell) are used interchangeably. Theamount of a tumor in an individual is the “tumor burden” which can bemeasured as the number, volume, or weight of the tumor.

The term “relapse” refers to the diagnosis of return, or signs andsymptoms of return, of a cancer after a period of improvement orremission.

“Remission,” is also referred to as “clinical remission,” and includesboth partial and complete remission. In partial remission, some, but notall, signs and symptoms of cancer have disappeared. In completeremission, all signs and symptoms of cancer have disappeared, althoughcancer still may be in the body.

“Refractory” refers to a cancer that is resistant to, or non-responsiveto, therapy with a particular therapeutic agent. A cancer can berefractory from the onset of treatment (i.e., non-responsive to initialexposure to the therapeutic agent), or as a result of developingresistance to the therapeutic agent, either over the course of a firsttreatment period or during a subsequent treatment period.

As used herein, the terms “individual” and “subject” are often usedinterchangeably and refer to any animal that exhibits a symptom ofcancer or other immune disorder that can be treated with thecompositions and methods contemplated elsewhere herein. Suitablesubjects (e.g., patients) include laboratory animals (such as mouse,rat, rabbit, or guinea pig), farm animals, and domestic animals or pets(such as a cat or dog). Non-human primates and, preferably, humanpatients, are included. Typical subjects include human patients thathave, have been diagnosed with, or are at risk or having, cancer oranother immune disorder.

As used herein, the term “patient” refers to a subject that has beendiagnosed with cancer or another immune disorder that can be treatedwith the compositions and methods disclosed elsewhere herein.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated.Treatment can involve optionally either the reduction of the disease orcondition, or the delaying of the progression of the disease orcondition, e.g., delaying tumor outgrowth. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition. It also refers to delaying the onset or recurrence of adisease or condition or delaying the occurrence or recurrence of thesymptoms of a disease or condition. As used herein, “prevention” andsimilar words also includes reducing the intensity, effect, symptomsand/or burden of a disease or condition prior to onset or recurrence ofthe disease or condition.

As used herein, the phrase “ameliorating at least one symptom of” refersto decreasing one or more symptoms of the disease or condition for whichthe subject is being treated. In particular embodiments, the disease orcondition being treated is a cancer, wherein the one or more symptomsameliorated include, but are not limited to, weakness, fatigue,shortness of breath, easy bruising and bleeding, frequent infections,enlarged lymph nodes, distended or painful abdomen (due to enlargedabdominal organs), bone or joint pain, fractures, unplanned weight loss,poor appetite, night sweats, persistent mild fever, and decreasedurination (due to impaired kidney function).

By “enhance” or “promote,” or “increase” or “expand” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a greater physiological response (i.e., downstream effects)compared to the response caused by either vehicle or a controlmolecule/composition. A measurable physiological response may include anincrease in T cell expansion, activation, persistence, cytokinesecretion, and/or an increase in cancer cell killing ability, amongothers apparent from the understanding in the art and the descriptionherein. An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the responseproduced by vehicle or a control composition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of composition contemplated herein to produce,elicit, or cause a lesser physiological response (i.e., downstreameffects) compared to the response caused by either vehicle or a controlmolecule/composition. A “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include a decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response(reference response) produced by vehicle, a control composition, or theresponse in a particular cell lineage.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” or “no substantial decrease” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a substantially similar or comparable physiological response(i.e., downstream effects) in a cell, as compared to the response causedby either vehicle, a control molecule/composition, or the response in aparticular cell lineage. A comparable response is one that is notsignificantly different or measurable different from the referenceresponse.

Additional definitions are set forth throughout this disclosure.

C. CD33 VHH DARICs

In particular embodiments, a DARIC receptor comprising an anti-CD33 VHHdomain that redirects cytotoxicity of immune effector cells towardcancer cells expressing CD33 is contemplated. As used herein, the terms“CD33 VHH DARIC receptor,” “anti-CD33 VHH DARIC receptor” “CD33 VHHDARIC,” or “anti-CD33 VHH DARIC” are used interchangeably and refer toone or more non-naturally occurring polypeptides that transduces animmunostimulatory signal in an immune effector cell upon exposure to atarget cell expressing full-length CD33 or a CD33 splice variant and amultimerizing agent or bridging factor, e.g., stimulating immuneeffector cell activity and function, increasing production and/orsecretion of proinflammatory cytokines. In preferred embodiments, a CD33VHH DARIC is a multi-chain chimeric receptor comprising a DARICsignaling component and a DARIC binding component comprising a VHHdomain that recognizes full-length CD33 and/or a CD33 splice variant.

In one embodiment, a DARIC signaling component and a DARIC bindingcomponent are expressed from the same cell. In another embodiment, aDARIC signaling component and a DARIC binding component are expressedfrom different cells. In a particular embodiment, a DARIC signalingcomponent is expressed from a cell and a DARIC binding component issupplied exogenously, as a polypeptide. In one embodiment, a DARICbinding component pre-loaded with a bridging factor is suppliedexogenously to a cell expressing a DARIC signaling component.

1. CD33 DARIC Signaling Component

The terms “DARIC signaling component,” CD33 DARIC signaling component,”“DARIC signaling polypeptide,” or “DARIC signaling polypeptide” are usedinterchangeably and refer to a polypeptide comprising one or moremultimerization domains, a transmembrane domain, and one or moreintracellular signaling domains. In particular embodiments, a DARICsignaling component comprises a multimerization domain, a transmembranedomain, a costimulatory domain and/or a primary signaling domain. Inparticular embodiments, a DARIC signaling component comprises a firstmultimerization domain, a first transmembrane domain, a firstcostimulatory domain and/or a primary signaling domain.

In particular embodiments, a DARIC signaling component comprises one ormore multimerization domains.

Illustrative examples of multimerization domains suitable for use inparticular CD33 DARIC signaling components contemplated herein include,but are not limited to, an FK506 binding protein (FKBP) polypeptide orvariants thereof, an FKBP-rapamycin binding (FRB) polypeptide orvariants thereof, a calcineurin polypeptide or variants thereof, acyclophilin polypeptide or variants thereof, a bacterial dihydrofolatereductase (DHFR) polypeptide or variants thereof, a PYR1-like 1 (PYL1)polypeptide or variants thereof and an abscisic acid insensitive 1(ABI1) polypeptide or variants thereof.

In particular embodiments, a CD33 DARIC signaling component comprises anFRB polypeptide.

In particular preferred embodiments, a CD33 DARIC signaling componentcomprises an FRB polypeptide comprising a T2098L mutation, or variantthereof. In certain preferred embodiments, a CD33 DARIC signalingcomponent comprises an FKBP12 polypeptide or variant thereof. In someembodiments, a CD33 VHH DARIC signaling component comprises a hingedomain.

Illustrative hinge domains suitable for use in a CD33 VHH DARICsignaling component described herein include the hinge region derivedfrom the extracellular regions of type 1 membrane proteins such as CD28,CD8a, and CD4, which may be wild-type hinge regions from these moleculesor may be altered.

In particular embodiments, a DARIC signaling component comprises atransmembrane domain.

In particular embodiments, a DARIC signaling component comprises a hingedomain and a transmembrane domain.

Illustrative examples of transmembrane domains suitable for use inparticular CD33 DARIC signaling components contemplated herein include,but are not limited to, the transmembrane region(s) of the alpha, beta,gamma, or delta chain of a T-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8a,CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86,CD 134, CD137, CD152, CD 154, amnionless (AMN), and programmed celldeath 1 (PDCD1). In a preferred embodiment, a CD33 DARIC signalingcomponent comprises a CD4 transmembrane domain. In a preferredembodiment, a CD33 DARIC signaling component comprises a CD8atransmembrane domain.

In particular embodiments, a DARIC signaling component comprises alinker that links the C-terminus of the transmembrane domain to theN-terminus of an intracellular signaling domain. In various preferredembodiments, a short oligo- or poly-peptide linker, preferably between1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length links thetransmembrane domain and an intracellular signaling domain. Aglycine-serine based linker provides a particularly suitable linker.

DARIC signaling components contemplated in particular embodiments hereincomprise one or more intracellular signaling domains. In one embodiment,a CD33 DARIC signaling component comprises one or more costimulatorysignaling domains and/or a primary signaling domain. In one embodiment,the intracellular signaling domain comprises an immunoreceptor tyrosineactivation motif (ITAM).

Illustrative examples of ITAM containing primary signaling domains thatare suitable for use in particular CD33 DARIC signaling componentscontemplated herein include, but are not limited to those derived fromFcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Inpreferred embodiments, a CD33 DARIC signaling component comprises a CD3ζprimary signaling domain and one or more costimulatory signalingdomains. The primary signaling and costimulatory signaling domains maybe linked in any order in tandem to the carboxyl terminus of thetransmembrane domain.

Illustrative examples of costimulatory domains suitable for use inparticular CD33 DARIC signaling components contemplated herein include,but are not limited to those domains isolated from the followingcostimulatory molecules: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain familymember 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83,CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptorassociated transmembrane adaptor 1 (TRAT1), TNFR2, TNF receptorsuperfamily member 14 (TNFRS14; HVEM), TNF receptor superfamily member18 (TNFRS18; GITR), TNF receptor superfamily member 25 (TNFRS25; DR3),and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).

In particular embodiments, a CD33 DARIC signaling component contemplatedherein comprises a signal peptide. Illustrative examples of signalpeptides suitable for use in particular CD33 DARIC signaling componentsinclude but are not limited to an IgG1 heavy chain signal polypeptide,an Igκ light chain signal polypeptide, a CD8a signal polypeptide, or ahuman GM-CSF receptor alpha signal polypeptide. In various preferredembodiments, a CD33 DARIC signaling component comprises a CD8a signalpolypeptide.

In particular embodiments, a CD33 DARIC signaling component comprisesone or more costimulatory signaling domains selected from the groupconsisting of CD28, CD137, and CD134. In particular embodiments, a CD33DARIC signaling component comprises one or more costimulatory signalingdomains selected from the group consisting of CD28, CD137, and CD134,and a CD3ζ primary signaling domain. In a particular embodiment, a CD33DARIC signaling component comprises a CD137 costimulatory domain and aCD3ζ primary signaling domain.

In a preferred embodiment, a CD33 DARIC signaling component comprises anFRB T2098L multimerization domain, a CD8a transmembrane domain, a CD137costimulatory domain and a CD3ζ primary signaling domain.

In preferred embodiments, a CD33 VHH DARIC signaling component comprisesan amino acid sequence set forth in SEQ ID NO 82.

2. CD33 DARIC Binding Component

A “DARIC binding component,” “DARIC binding polypeptide,” “CD33 VHHDARIC binding component,” or “CD33 VHH DARIC binding polypeptide” areused interchangeably and refer to a polypeptide comprising an anti-CD33VHH domain, and one or more multimerization domains. In particularembodiments, the CD33 VHH DARIC binding component comprises an anti-CD33VHH domain, a multimerization domain and a transmembrane domain. Inparticular embodiments, the CD33 VHH DARIC binding component comprisesan anti-CD33 VHH domain, a second multimerization domain, and a secondtransmembrane domain. In other particular embodiments, the CD33 VHHDARIC binding component comprises an anti-CD33 VHH domain, amultimerization domain, a transmembrane domain and one or moreintracellular signaling domains. In particular embodiments, the CD33 VHHDARIC binding component comprises an anti-CD33 VHH domain, a secondmultimerization domain, a second transmembrane domain, and a secondcostimulatory domain.

In particular embodiments, the CD33 VHH DARIC binding componentcomprises one or more anti-CD33 VHH domains.

In particular preferred embodiments, the anti-CD33 VHH domain is ahumanized camelid VHH. In particular embodiments, the anti-CD33 VHHdomain is a humanized camelid VHH that binds one or more epitopes offull-length CD33 (e.g., SEQ ID NO: 1) or one or more epitopes of a CD33splice variant. In particular embodiments, the anti-CD33 VHH domain is ahumanized camelid VHH that binds the same one or more epitopes displayedon both full-length CD33 and a CD33 splice variant.

In particular preferred embodiments, the anti-CD33 VHH domain is ahumanized camelid VHH comprising amino acid sequence set forth in anyone of SEQ ID NOs: 3-6, 10-11, 13-16, and 20-21. In certain preferredembodiments, the anti-CD33 VHH domain is a humanized camelid VHHcomprising amino acid sequence set forth in SEQ ID NO: 10. In certainpreferred embodiments, the anti-CD33 VHH domain is a humanized camelidVHH comprising amino acid sequence set forth in SEQ ID NO: 20.

In particular embodiments, a DARIC binding component comprises one ormore multimerization domains.

Illustrative examples of multimerization domains suitable for use inparticular CD33 VHH DARIC binding components contemplated hereininclude, but are not limited to, an FKBP polypeptide or variantsthereof, an FRB polypeptide or variants thereof, a calcineurinpolypeptide or variants thereof, a cyclophilin polypeptide or variantsthereof, a DHFR polypeptide or variants thereof, a PYL1 polypeptide orvariants thereof and an ABI1 polypeptide or variants thereof.

In particular embodiments, a CD33 VHH DARIC binding component comprisesan FRB polypeptide or variant thereof and a DARIC signaling componentcomprises an FKBP polypeptide or variant thereof. In a preferredembodiment, a CD33 VHH DARIC binding component comprises an FRBpolypeptide comprising a T2098L mutation, or variant thereof and a DARICsignaling component comprises an FKBP12 polypeptide or variant thereof.

In particular embodiments, a CD33 VHH DARIC binding component comprisesan FKBP polypeptide or variant thereof and a DARIC signaling componentcomprises an FRB polypeptide, or variant thereof. In a preferredembodiment, a CD33 VHH DARIC binding component comprises an FKBP12polypeptide, or variant thereof and a DARIC signaling componentcomprises an FRB polypeptide comprising a T2098L mutation, or variantthereof.

In some embodiments, a CD33 VHH DARIC binding component comprises ahinge domain.

Illustrative hinge domains suitable for use in a CD33 VHH DARIC bindingcomponent described herein include the hinge region derived from theextracellular regions of type 1 membrane proteins such as CD28, CD8a,and CD4, which may be wild-type hinge regions from these molecules ormay be altered.

In particular embodiments, a DARIC binding component comprises atransmembrane domain. In particular embodiments, a DARIC bindingcomponent comprises a hinge domain and a transmembrane domain. In oneembodiment, the transmembrane domain may be the same as thetransmembrane domain used in the DARIC signaling component. In oneembodiment, the transmembrane domain may be different from thetransmembrane domain used in the DARIC signaling component.

Illustrative examples of transmembrane domains suitable for use inparticular CD33 VHH DARIC binding components contemplated hereininclude, but are not limited to, the transmembrane region(s) of thealpha, beta, gamma, or delta chain of a T-cell receptor, CD3ε, CD3ζ,CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64,CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, amnionless (AMN), andprogrammed cell death 1 (PDCD1). In a preferred embodiment, a CD33 DARICbinding component comprises a CD8a transmembrane domain. In a preferredembodiment, a CD33 VHH DARIC binding component comprises a CD4transmembrane domain.

In various preferred embodiments, a short oligo- or poly-peptide linker,preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inlength links the transmembrane domain and the intracellular signalingdomain. A glycine-serine based linker provides a particularly suitablelinker.

DARIC binding components contemplated in particular embodiments hereindo not comprise one or more intracellular signaling domains.

In other particular embodiments, CD33 VHH DARIC binding componentscontemplated herein comprise one or more intracellular signalingdomains. In preferred embodiments, wherein the CD33 VHH DARIC bindingcomponent comprises one or more intracellular signaling domains, thosedomains are different that the intracellular signaling domains presentin the cognate CD33 DARIC signaling component. In one embodiment, a CD33VHH DARIC binding component comprises a costimulatory signaling domain.

Illustrative examples of costimulatory domains suitable for use inparticular CD33 VHH DARIC binding components contemplated hereininclude, but are not limited to those domains isolated from thefollowing costimulatory molecules: Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitmentdomain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS),DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cellsfamily member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD(SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1),TNFR2, TNF receptor superfamily member 14 (TNFRS14; HVEM), TNF receptorsuperfamily member 18 (TNFRS18; GITR), TNF receptor superfamily member25 (TNFRS25; DR3), and zeta chain of T cell receptor associated proteinkinase 70 (ZAP70). In preferred embodiments, the costimulatory domain isderived, obtained, or isolated from TNFR2 or OX40.

In particular embodiments, a DARIC binding component contemplated hereincomprises a signal peptide. Illustrative examples of signal peptidessuitable for use in particular CD33 VHH DARIC binding components includebut are not limited to an IgG1 heavy chain signal polypeptide, an Igκlight chain signal polypeptide, a CD8a signal polypeptide, or a humanGM-CSF receptor alpha signal polypeptide. In various preferredembodiments, a CD33 VHH DARIC binding component comprises a CD8a signalpolypeptide.

In particular embodiments, a CD33 VHH DARIC binding component comprisesa VHH domain that binds to CD33, an FKBP12 multimerization domain, and aCD4 transmembrane domain and optionally, a costimulatory domain.

In certain embodiments, a CD33 VHH DARIC binding component comprises aVHH that binds to CD33, and an FKBP12 multimerization domain.

In some embodiments, a CD33 VHH DARIC binding component comprises a VHHdomain comprising the amino acid sequence set forth in any one of SEQ IDNOs: 2-21, an FKBP12 multimerization domain, and a CD4 transmembranedomain and optionally, a costimulatory domain.

In some embodiments, a CD33 VHH DARIC binding component comprises a VHHdomain comprising the amino acid sequence set forth in SEQ ID NO: 10 orSEQ ID NO: 20, an FKBP12 multimerization domain, and a CD4 transmembranedomain and optionally, a costimulatory domain.

In some embodiments, a CD33 VHH DARIC binding component comprises a VHHdomain comprising the amino acid sequence set forth in any one of SEQ IDNOs: 2-21, and an FKBP12 multimerization domain.

In some embodiments, a CD33 VHH DARIC binding component comprises a VHHdomain comprising the amino acid sequence set forth in SEQ ID NO: 10 orSEQ ID NO: 20, and an FKBP12 multimerization domain.

In some embodiments, a CD33 VHH DARIC binding component comprises theamino acid sequence set forth in any one of SEQ ID NOs: 22-31.

In some embodiments, a CD33 VHH DARIC binding component comprises theamino acid sequence set forth in SEQ ID NO: 30.

3. Bridging Factor

Bridging factors contemplated in particular embodiments herein, mediateor promote the association of a CD33 DARIC signaling component with aCD33 VHH DARIC binding component through multimerization domains in therespective components. A bridging factor associates with and is disposedbetween the multimerization domains to promote association of a CD33DARIC signaling component and a CD33 VHH DARIC binding component. In thepresence of a bridging factor, the CD33 VHH DARIC binding component andthe CD33 DARIC signaling component associate and initiate immuneeffector cell activity against a target cell when the CD33 VHH DARICbinding component binds CD33 expressed on the target cell. In theabsence of a bridging factor, the CD33 VHH DARIC binding component doesnot associate with the CD33 DARIC signaling component and the CD33 VHHDARIC is inactive.

In particular embodiments, a CD33 DARIC signaling component and a CD33VHH DARIC binding component comprise a cognate pair of multimerizationdomains selected from the group consisting of: FKBP and FKBP12-rapamycinbinding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP andbacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin,and PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).

In certain embodiments, the multimerization domains of CD33 VHH DARICsignaling and binding components associate with a bridging factorselected from the group consisting of: rapamycin or a rapalog thereof,coumermycin or a derivative thereof, gibberellin or a derivativethereof, abscisic acid (ABA) or a derivative thereof, methotrexate or aderivative thereof, cyclosporin A or a derivative thereof,FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim(Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or aderivative thereof.

In particular embodiments, a CD33 DARIC signaling component and a CD33VHH DARIC binding component comprise one or more FRB and/or FKBPmultimerization domains or variants thereof. In certain embodiments, aCD33 DARIC signaling component comprises an FRB multimerization domainor variant thereof and a CD33 VHH DARIC binding component comprises anFKBP multimerization domain or variant thereof. In particular preferredembodiments, a CD33 DARIC signaling component comprises an FRB T2098Lmultimerization domain or variant thereof and a CD33 VHH DARIC bindingcomponent comprises an FKBP12 or FKBP12 F36V multimerization domains orvariant thereof.

Illustrative examples of bridging factors suitable for use in particularembodiments contemplated herein include, but are not limited to, AP1903,AP20187, AP21967 (also known as C-16-(S)-7-methylindolerapamycin),everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, and zotarolimus. In particular preferredembodiments, the bridging factor is AP21967. In certain preferredembodiments, the bridging factor is a non-immunosuppressive dose ofsirolimus (rapamycin).

D. Anti-CD33 Chimeric Antigen Receptors

In particular embodiments, immune effector cells contemplated hereincomprise an anti-CD33 VHH CAR. Chimeric antigen receptors (CARs) aremolecules that combine antibody-based specificity for a target antigen(e.g., tumor antigen) with a T cell receptor-activating intracellulardomain to generate a chimeric protein that exhibits a specificanti-tumor cellular immune activity. As used herein, the term,“chimeric,” describes being composed of parts of different proteins orDNAs from different origins.

In particular embodiments, T cells are engineered by introducing apolynucleotide encoding an anti-CD33 VHH CAR.

In particular embodiments, T cells are engineered by introducing avector comprising a polynucleotide encoding an anti-CD33 VHH CAR.

In various embodiments, an anti-CD33 CAR comprises a VHH domain thatbinds CD33, a transmembrane domain and one or more intracellularsignaling domains. The main characteristic of CARs is their ability toredirect immune effector cell specificity, thereby triggeringproliferation, cytokine production, phagocytosis or production ofmolecules that can mediate cell death of the target antigen expressingcell in a major histocompatibility (MHC) independent manner, exploitingthe cell specific targeting abilities of monoclonal antibodies, solubleligands or cell specific coreceptors.

In some embodiments, an anti-CD33 VHH CAR comprises a spacer domain. Inparticular embodiments, the spacer domain comprises the CH2 and CH3 ofIgG1, IgG4, or IgD.

Illustrative hinge domains suitable for use in the anti-CD33 VHH CARSdescribed herein include the hinge region derived from the extracellularregions of type 1 membrane proteins such as CD28, CD8a, and CD4, whichmay be wild-type hinge regions from these molecules or may be altered.In another embodiment, the hinge domain comprises a CD8a hinge region.

The transmembrane (TM) domain of the CAR fuses the extracellular bindingportion and intracellular signaling domain and anchors the CAR to theplasma membrane of the immune effector cell. The TM domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource.

Illustrative TM domains may be derived from (i.e., comprise at least thetransmembrane region(s) of the alpha, beta, gamma, or delta chain of aT-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PDCD1.

In one embodiment, an anti-CD33 VHH CAR comprises a TM domain derivedfrom CD8α. In another embodiment, a CAR contemplated herein comprises aTM domain derived from CD8α and a short oligo- or polypeptide linker,preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inlength that links the TM domain and the intracellular signaling domainof the CAR. A glycine-serine linker provides a particularly suitablelinker.

In preferred embodiments, an anti-CD33 VHH CAR comprises anintracellular signaling domain that comprises one or more costimulatorysignaling domains and a primary signaling domain.

Primary signaling domains that act in a stimulatory manner may containsignaling motifs which are known as immunoreceptor tyrosine-basedactivation motifs or ITAMs.

Illustrative examples of ITAM containing primary signaling domainssuitable for use in anti-CD33 VHH CARs contemplated in particularembodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε,CD3ζ, CD22, CD79a, CD79b, and CD66d. In particular preferredembodiments, a CAR comprises a CD3ζ primary signaling domain and one ormore costimulatory signaling domains. The intracellular primarysignaling and costimulatory signaling domains may be linked in any orderin tandem to the carboxyl terminus of the transmembrane domain.

In particular embodiments, an anti-CD33 VHH CAR comprises one or morecostimulatory signaling domains to enhance the efficacy and expansion ofT cells expressing CAR receptors.

Illustrative examples of such costimulatory molecules suitable for usein anti-CD33 VHH CARs contemplated in particular embodiments include,but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM),CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT,SLP76, TRAT1, TNFR2, and ZAP70. In one embodiment, a CAR comprises oneor more costimulatory signaling domains selected from the groupconsisting of CD28, CD137, and CD134, and a CD3ζ primary signalingdomain.

In various embodiments, the anti-CD33 VHH CAR comprises: a VHH thatbinds CD33; a transmembrane domain isolated from a polypeptide selectedfrom the group consisting of: CD4, CD8a, CD154, and PD-1; one or moreintracellular costimulatory signaling domains isolated from apolypeptide selected from the group consisting of: CD28, CD134, andCD137; and a signaling domain isolated from a polypeptide selected fromthe group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, and CD66d.

In various embodiments, the anti-CD33 VHH CAR comprises: a VHH thatbinds CD33; a transmembrane domain isolated from a polypeptide selectedfrom the group consisting of: CD4, CD8a, CD154, and PD-1; one or moreintracellular costimulatory signaling domains isolated from apolypeptide selected from the group consisting of: CD28, CD134, andCD137; and a signaling domain isolated from a polypeptide selected fromthe group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, and CD66d.

In preferred embodiments, an anti-CD33 VHH CAR comprises a VHH thatcomprises an amino acid sequence set forth in any one of SEQ ID NOs:2-21, a CD8a hinge domain, a CD8a transmembrane domain, a 4-1BBcostimulatory domain, and a CD3ζ primary signaling domain.

In preferred embodiments, an anti-CD33 VHH CAR comprises a VHH thatcomprises an amino acid sequence set forth in SEQ ID NO: 10, a CD8ahinge domain, a CD8a transmembrane domain, a 4-1BB costimulatory domain,and a CD3ζ primary signaling domain.

In particular embodiments, an anti-CD33 VHH CAR comprises a sequence setforth in any one of SEQ ID NOs: 62-81.

In particular embodiments, an anti-CD33 VHH CAR comprises a sequence setforth in SEQ ID NO: 70 or SEQ ID NO: 80.

E. Polypeptides

Various polypeptides are contemplated herein, including, but not limitedto, CD33 VHH DARICs, CD33 VHH DARIC binding components, CD33 DARICsignaling components, anti-CD33 VHH CARS, and fragments thereof. Inpreferred embodiments, a polypeptide comprises an amino acid sequenceset forth in any one of SEQ ID NOs: 2-82. “Polypeptide,” “peptide” and“protein” are used interchangeably, unless specified to the contrary,and according to conventional meaning, i.e., as a sequence of aminoacids. In one embodiment, a “polypeptide” includes fusion polypeptidesand other variants. Polypeptides can be prepared using any of a varietyof well-known recombinant and/or synthetic techniques. Polypeptides arenot limited to a specific length, e.g., they may comprise a full-lengthprotein sequence, a fragment of a full-length protein, or a fusionprotein, and may include post-translational modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. In particular preferredembodiments, fusion polypeptides, polypeptides, fragments and othervariants thereof are prepared, obtained, or isolated from one or morehuman polypeptides.

An “isolated peptide” or an “isolated polypeptide” and the like, as usedherein, refer to in vitro isolation and/or purification of a peptide orpolypeptide molecule from a cellular environment, and from associationwith other components of the cell, i.e., it is not significantlyassociated with in vivo substances. In particular embodiments, anisolated polypeptide is a synthetic polypeptide, a semi-syntheticpolypeptide, or a polypeptide obtained or derived from a recombinantsource.

Polypeptides include “polypeptide variants.” Polypeptide variants maydiffer from a naturally occurring polypeptide in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring, e.g., a splice variant, or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences. For example, in particular embodiments, it may bedesirable to improve the binding affinity and/or other biologicalproperties of a polypeptide by introducing one or more substitutions,deletions, additions and/or insertions the polypeptide. In particularembodiments, polypeptides include polypeptides having at least about65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of thereference sequences contemplated herein, typically where the variantmaintains at least one biological activity of the reference sequence. Inparticular embodiments, the biological activity is binding affinity. Inparticular embodiments, the biological activity is cytolytic activity.

Polypeptide variants include biologically active “polypeptidefragments.” Illustrative examples of biologically active polypeptidefragments include anti-CD33 VHH domains, intracellular signalingdomains, and the like. As used herein, the term “biologically activefragment” or “minimal biologically active fragment” refers to apolypeptide fragment that retains at least 100%, at least 90%, at least80%, at least 70%, at least 60%, at least 50%, at least 40%, at least30%, at least 20%, at least 10%, or at least 5% of the naturallyoccurring polypeptide activity. In certain embodiments, a polypeptidefragment can comprise an amino acid chain at least 5 to about 1700 aminoacids long. It will be appreciated that in certain embodiments,fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700 or more amino acids long.

In particular embodiments, the polypeptides set forth herein maycomprise one or more amino acids denoted as “X” or “Xaa,” which are usedinterchangeably. “X” if present in an amino acid SEQ ID NO, refers toany one or more amino acids. In particular embodiments, SEQ ID NOsdenoting a fusion protein comprise a sequence of continuous X residuesthat cumulatively represent any amino acid sequence. In particularembodiments, “XX” represent any two amino acid combination. In certainembodiments, “XX” represents two serines, SS. In certain embodiments,“XX” represents any two amino acid combination that reducesimmunogenicity.

In preferred embodiments, “XX” represents the amino acids KP.

As noted above, polypeptides may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a reference polypeptide can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987,Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif., 1987) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al., (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found, Washington, D.C.).

In certain embodiments, a polypeptide variant comprises one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Modifications may be madein the structure of the polynucleotides and polypeptides contemplated inparticular embodiments and still obtain a functional molecule thatencodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variantpolypeptide, one skilled in the art, for example, can change one or moreof the codons of the encoding DNA sequence, e.g., according to Table 1.

TABLE 1 Amino Acid Codons One Three letter letter Amino Acids code codeCodons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Asparticacid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine F Phe UUCUUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU Isoleucine IIso AUA AUC AUU Lysine K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC CUGCUU Methionine M Met AUG Asparagine N Asn AAC AAU Proline P Pro CCA CCCCCG CCU Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGUSerine S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACUValine V Val GUA GUC GUG GUU Tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs well known in the art, such as DNASTAR, DNAStrider, Geneious, Mac Vector, or Vector NTI software. Preferably, aminoacid changes in the protein variants disclosed herein are conservativeamino acid changes, i.e., substitutions of similarly charged oruncharged amino acids. A conservative amino acid change involvessubstitution of one of a family of amino acids which are related intheir side chains. Naturally occurring amino acids are generally dividedinto four families: acidic (aspartate, glutamate), basic (lysine,arginine, histidine), non-polar (alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), and uncharged polar(glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine)amino acids. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In a peptide or protein,suitable conservative substitutions of amino acids are known to those ofskill in this art and generally can be made without altering abiological activity of a resulting molecule. Those of skill in this artrecognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. Molecular Biology of theGene, 4th Edition, 1987, The Benj amin/Cummings Pub. Co., p. 224).

In one embodiment, where expression of two or more polypeptides isdesired, the polynucleotide sequences encoding them can be separated byan IRES sequence or a polynucleotide sequence encoding a ribosomal skipsequence as disclosed elsewhere herein.

Polypeptides contemplated in particular embodiments include fusionpolypeptides. In particular embodiments, fusion polypeptides andpolynucleotides encoding fusion polypeptides are provided. Fusionpolypeptides and fusion proteins refer to a polypeptide having at leasttwo, three, four, five, six, seven, eight, nine, or ten polypeptidesegments. In preferred embodiments, a fusion polypeptide comprises oneor more CD33 VHH DARIC components. In other preferred embodiments, thefusion polypeptide comprises one or more CD33 VHH DARICs.

In another embodiment, two or more CD33 VHH DARIC components and/orother polypeptides can be expressed as a fusion protein that comprisesone or more self-cleaving peptide sequences between the polypeptides asdisclosed elsewhere herein.

In particular embodiments, a fusion polypeptide comprises a CD33 DARICsignaling component, a self-cleaving polypeptide sequence or ribosomalskip sequence, and a CD33 VHH DARIC binding component.

In particular embodiments, a fusion polypeptide comprises a CD33 DARICsignaling component, a self-cleaving polypeptide sequence or ribosomalskip sequence, a CD33 VHH DARIC binding component, another self-cleavingpolypeptide sequence or ribosomal skip sequence, and another DARICbinding component that is directed against another target antigen.

Fusion polypeptides can comprise one or more polypeptide domains orsegments including, but are not limited to signal peptides, cellpermeable peptide domains (CPP), binding domains, signaling domains,etc., epitope tags (e.g., maltose binding protein (“MBP”), glutathione Stransferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptidelinkers, and polypeptide cleavage signals. Fusion polypeptides aretypically linked C-terminus to N-terminus, although they can also belinked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminusto C-terminus. In particular embodiments, the polypeptides of the fusionprotein can be in any order. Fusion polypeptides or fusion proteins canalso include conservatively modified variants, polymorphic variants,alleles, mutants, subsequences, and interspecies homologs, so long asthe desired activity of the fusion polypeptide is preserved. Fusionpolypeptides may be produced by chemical synthetic methods or bychemical linkage between the two moieties or may generally be preparedusing other standard techniques. Ligated DNA sequences comprising thefusion polypeptide are operably linked to suitable transcriptional ortranslational control elements as disclosed elsewhere herein.

Fusion polypeptides may optionally comprise one or more linkers that canbe used to link the one or more polypeptides or domains within apolypeptide. A peptide linker sequence may be employed to separate anytwo or more polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its appropriate secondary and tertiarystructures so as to allow the polypeptide domains to exert their desiredfunctions. Such a peptide linker sequence is incorporated into thefusion polypeptide using standard techniques in the art. Suitablepeptide linker sequences may be chosen based on the following factors:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes. In particular embodiments, preferredpeptide linker sequences contain Gly, Asn and Ser residues. Other nearneutral amino acids, such as Thr and Ala may also be used in the linkersequence. Amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphyet al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos.4,935,233 and 4,751,180. Linker sequences are not required when aparticular fusion polypeptide segment contains non-essential N-terminalamino acid regions that can be used to separate the functional domainsand prevent steric interference. In particular embodiments, preferredlinkers are typically flexible amino acid subsequences which aresynthesized as part of a recombinant fusion protein. Linker polypeptidescan be between 1 and 200 amino acids in length, between 1 and 100 aminoacids in length, or between 1 and 50 amino acids in length, includingall integer values in between.

Exemplary polypeptide cleavage signals include polypeptide cleavagerecognition sites such as protease cleavage sites, nuclease cleavagesites (e.g., rare restriction enzyme recognition sites, self-cleavingribozyme recognition sites), and self-cleaving viral oligopeptides (seedeFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., in Ryan et al., 1997. J. Gener. Virol.78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus Ma proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.Due to its high cleavage stringency, TEV (tobacco etch virus) proteasecleavage sites are preferred in one embodiment, e.g., EXXYXQ(G/S) (SEQID NO: 95), for example, ENLYFQG (SEQ ID NO: 96) and ENLYFQS (SEQ ID NO:97), wherein X represents any amino acid (cleavage by TEV occurs betweenQ and G or Q and S).

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving peptide or ribosomal skipping sequence.

Illustrative examples of ribosomal skipping sequences include, but arenot limited to: a 2A or 2A-like site, sequence or domain (Donnelly etal., 2001. J. Gen. Virol. 82:1027-1041). In a particular embodiment, theviral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide,or a cardiovirus 2A peptide.

In one embodiment, the viral 2A peptide is selected from the groupconsisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, anequine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV)2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2Apeptide, and an encephalomyocarditis virus 2A peptide.

Illustrative examples of 2A sites are provided in Table 2.

TABLE 2 SEQ ID NO: 98 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 99ATNFSLLKQAGDVEENPGP SEQ ID NO: 100 LLKQAGDVEENPGP SEQ ID NO: 101GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 102 EGRGSLLTCGDVEENPGP SEQ ID NO: 103LLTCGDVEENPGP SEQ ID NO: 104 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 105QCTNYALLKLAGDVESNPGP SEQ ID NO: 106 LLKLAGDVESNPGP SEQ ID NO: 107GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 108 VKQTLNFDLLKLAGDVESNPGPSEQ ID NO: 109 LLKLAGDVESNPGP SEQ ID NO: 110 LLNFDLLKLAGDVESNPGPSEQ ID NO: 111 TLNFDLLKLAGDVESNPGP SEQ ID NO: 112 LLKLAGDVESNPGPSEQ ID NO: 113 NFDLLKLAGDVESNPGP SEQ ID NO: 114 QLLNFDLLKLAGDVESNPGPSEQ ID NO: 115 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 116VTELLYRMKRAETYCPRPLLAIHPTEARHKQKI VAPVKQT SEQ ID NO: 117LNFDLLKLAGDVESNPGP SEQ ID NO: 118 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 119 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In preferred embodiments, a polypeptide or fusion polypeptide comprisesone or more CD33 VHH DARIC components, CD33 VHH DARICs, or anti-CD33 VHHCAR.

In preferred embodiments, a fusion polypeptide comprises a CD33 DARICsignaling component and a CD33 VHH DARIC binding component separated bya self-cleaving polypeptide sequence.

In particular embodiments, a fusion polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 32-61. In particular embodiments, afusion polypeptide comprises the sequence set forth in any one of SEQ IDNOs: 40, 50, or 60.

In particular embodiments, a fusion polypeptide comprises a CD33 DARICsignaling component comprising an FRB T2098L multimerization domain, aCD8a transmembrane domain, a CD137 costimulatory domain and a CD3ζprimary signaling domain; a viral self-cleaving 2A polypeptide; and aCD33 VHH DARIC binding component comprising an anti-CD33 VHH, an FKBP12multimerization domain polypeptide, and a CD4 transmembrane domain.

In particular embodiments, a fusion polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 32-41. In particular embodiments, afusion polypeptide comprises the sequence set forth in SEQ ID NO: 40.

In particular embodiments, a fusion polypeptide comprises a CD33 DARICsignaling component comprising an FRB T2098L multimerization domain, aCD8a transmembrane domain, a CD137 costimulatory domain and a CD3ζprimary signaling domain; a viral self-cleaving 2A polypeptide; and ananti-CD33 VHH, a CD4 transmembrane domain, and optionally a CD27, CD28,TNFRS14, TNFRS18, TNFRS25, OX40 or TNFR2 costimulatory domain.

In particular embodiments, a fusion polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 42-51. In particular embodiments, afusion polypeptide comprises the sequence set forth in SEQ ID NO: 50.

In particular embodiments, a fusion polypeptide comprises the sequenceset forth in any one of SEQ ID NOs: 52-61. In particular embodiments, afusion polypeptide comprises the sequence set forth in SEQ ID NO: 60.

F. Polynucleotides

In particular embodiments, polynucleotides encoding CD33 VHH DARICs,CD33 VHH DARIC binding components, CD33 DARIC signaling components,anti-CD33 VHH CARs and fragments thereof are provided. As used herein,the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleicacid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids. Polynucleotidesmay be single-stranded or double-stranded and either recombinant,synthetic, or isolated. Polynucleotides include, but are not limited to:pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, synthetic RNA,synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA(cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to apolymeric form of nucleotides of at least 5, at least 10, at least 15,at least 20, at least 25, at least 30, at least 40, at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 1000, at least 5000, at least 10000, or at least 15000 or morenucleotides in length, either ribonucleotides or deoxyribonucleotides ora modified form of either type of nucleotide, as well as allintermediate lengths. It will be readily understood that “intermediatelengths,” in this context, means any length between the quoted values,such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201,202, 203, etc. In particular embodiments, polynucleotides or variantshave at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to a reference sequence.

As used herein, “isolated polynucleotide” refers to a polynucleotidethat has been purified from the sequences which flank it in anaturally-occurring state, e.g., a DNA fragment that has been removedfrom the sequences that are normally adjacent to the fragment. Inparticular embodiments, an “isolated polynucleotide” also refers to acomplementary DNA (cDNA), a recombinant DNA, or other polynucleotidethat does not exist in nature and that has been made by the hand of man.In particular embodiments, an isolated polynucleotide is a syntheticpolynucleotide, a semi-synthetic polynucleotide, or a polynucleotideobtained or derived from a recombinant source.

In various embodiments, a polynucleotide comprises an mRNA encoding apolypeptide contemplated herein. In certain embodiments, the mRNAcomprises a cap, one or more nucleotides, and a poly(A) tail.

In particular embodiments, polynucleotides encoding one or more CD33 VHHDARIC components may be codon-optimized. As used herein, the term“codon-optimized” refers to substituting codons in a polynucleotideencoding a polypeptide in order to increase the expression, stabilityand/or activity of the polypeptide. Factors that influence codonoptimization include, but are not limited to one or more of: (i)variation of codon biases between two or more organisms or genes orsynthetically constructed bias tables, (ii) variation in the degree ofcodon bias within an organism, gene, or set of genes, (iii) systematicvariation of codons including context, (iv) variation of codonsaccording to their decoding tRNAs, (v) variation of codons according toGC %, either overall or in one position of the triplet, (vi) variationin degree of similarity to a reference sequence for example a naturallyoccurring sequence, (vii) variation in the codon frequency cutoff,(viii) structural properties of mRNAs transcribed from the DNA sequence,(ix) prior knowledge about the function of the DNA sequences upon whichdesign of the codon substitution set is to be based, (x) systematicvariation of codon sets for each amino acid, and/or (xi) isolatedremoval of spurious translation initiation sites.

As used herein the term “nucleotide” refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are understood to include natural bases, and a wide varietyof art-recognized modified bases. Such bases are generally located atthe position of a nucleotide sugar moiety. Nucleotides generallycomprise a base, sugar and a phosphate group. In ribonucleic acid (RNA),the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is adeoxyribose, i.e., a sugar lacking a hydroxyl group that is present inribose.

Illustrative examples of polynucleotides include, but are not limitedto, polynucleotides encoding polypeptides set forth in SEQ ID NOs: 2-82.

In various illustrative embodiments, polynucleotides contemplated hereininclude, but are not limited to polynucleotides encoding one or moreCD33 VHH DARIC components, CD33 VHH DARIC receptors, anti-CD33 VHH CARS,fusion polypeptides, and expression vectors, viral vectors, and transferplasmids comprising polynucleotides contemplated herein.

As used herein, the terms “polynucleotide variant” and “variant” and thelike refer to polynucleotides displaying substantial sequence identitywith a reference polynucleotide sequence or polynucleotides thathybridize with a reference sequence under stringent conditions that aredefined hereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletion,substitution, or modification of at least one nucleotide. Accordingly,the terms “polynucleotide variant” and “variant” include polynucleotidesin which one or more nucleotides have been added or deleted, ormodified, or replaced with different nucleotides. In this regard, it iswell understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains thebiological function or activity of the reference polynucleotide.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequenceidentity to any of the reference sequences described herein.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art, such that theiroverall length may vary considerably. It is therefore contemplated thata polynucleotide fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated, expressed and/or deliveredusing any of a variety of well-established techniques known andavailable in the art. In order to express a desired polypeptide, anucleotide sequence encoding the polypeptide, can be inserted intoappropriate vector.

Illustrative examples of vectors include, but are not limited toplasmid, autonomously replicating sequences, and transposable elements,e.g., Sleeping Beauty, PiggyBac.

Additional Illustrative examples of vectors include, without limitation,plasmids, phagemids, cosmids, artificial chromosomes such as yeastartificial chromosome (YAC), bacterial artificial chromosome (BAC), orP1-derived artificial chromosome (PAC), bacteriophages such as lambdaphage or M13 phage, and animal viruses.

Illustrative examples of viruses useful as vectors include, withoutlimitation, retrovirus (including lentivirus), adenovirus,adeno-associated virus, herpesvirus (e.g., herpes simplex virus),poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).

Illustrative examples of expression vectors include, but are not limitedto, pClneo vectors (Promega) for expression in mammalian cells;pLenti4N5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen)for lentivirus-mediated gene transfer and expression in mammalian cells.In particular embodiments, coding sequences of polypeptides disclosedherein can be ligated into such expression vectors for the expression ofthe polypeptides in mammalian cells.

In particular embodiments, the vector is an episomal vector or a vectorthat is maintained extrachromosomally. As used herein, the term“episomal” refers to a vector that is able to replicate withoutintegration into host's chromosomal DNA and without gradual loss from adividing host cell also meaning that said vector replicatesextrachromosomally or episomally.

“Expression control sequences,” “control elements,” or “regulatorysequences” present in an expression vector are those non-translatedregions of the vector including an origin of replication, selectioncassettes, promoters, enhancers, translation initiation signals (ShineDalgarno sequence or Kozak sequence) introns, a polyadenylationsequence, 5′ and 3′ untranslated regions, all of which interact withhost cellular proteins to carry out transcription and translation. Suchelements may vary in their strength and specificity. Depending on thevector system and host utilized, any number of suitable transcriptionand translation elements, including ubiquitous promoters and induciblepromoters may be used.

In particular embodiments, a polynucleotide comprises a vector,including but not limited to expression vectors and viral vectors. Avector may comprise one or more exogenous, endogenous, or heterologouscontrol sequences such as promoters and/or enhancers. An “endogenouscontrol sequence” is one which is naturally linked with a given gene inthe genome. An “exogenous control sequence” is one which is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter. A “heterologous controlsequence” is an exogenous sequence that is from a different species thanthe cell being genetically manipulated. A “synthetic” control sequencemay comprise elements of one more endogenous and/or exogenous sequences,and/or sequences determined in vitro or in silico that provide optimalpromoter and/or enhancer activity for the particular therapy.

The term “promoter” as used herein refers to a recognition site of apolynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNApolymerase initiates and transcribes polynucleotides operably linked tothe promoter. In particular embodiments, promoters operative inmammalian cells comprise an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated and/oranother sequence found 70 to 80 bases upstream from the start oftranscription, a CNCAAT region where N may be any nucleotide.

The term “enhancer” refers to a segment of DNA which contains sequencescapable of providing enhanced transcription and in some instances canfunction independent of their orientation relative to another controlsequence. An enhancer can function cooperatively or additively withpromoters and/or other enhancer elements. The term “promoter/enhancer”refers to a segment of DNA which contains sequences capable of providingboth promoter and enhancer functions.

The term “operably linked”, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. In one embodiment, the term refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, and/or enhancer) and a second polynucleotidesequence, e.g., a polynucleotide-of-interest, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

As used herein, the term “constitutive expression control sequence”refers to a promoter, enhancer, or promoter/enhancer that continually orcontinuously allows for transcription of an operably linked sequence. Aconstitutive expression control sequence may be a “ubiquitous” promoter,enhancer, or promoter/enhancer that allows expression in a wide varietyof cell and tissue types or a “cell specific,” “cell type specific,”“cell lineage specific,” or “tissue specific” promoter, enhancer, orpromoter/enhancer that allows expression in a restricted variety of celland tissue types, respectively.

Illustrative ubiquitous expression control sequences suitable for use inparticular embodiments include, but are not limited to, acytomegalovirus (CMV) immediate early promoter, a viral simian virus 40(SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV)LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters fromvaccinia virus, an elongation factor 1-alpha (EF1a) promoter, earlygrowth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL),Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translationinitiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein70 kDa (HSP70), β-kinesin ((3-KIN), the human ROSA 26 locus (Irions etal., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, a β-actin promoter and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, d1587rev primer-binding site substituted (MND) U3 promoter(Haas et al. Journal of Virology. 2003; 77(17): 9439-9450).

In one embodiment, a vector comprises an MNDU3 promoter.

In one embodiment, a vector comprises an EF1a promoter comprising thefirst intron of the human EF1a gene.

In one embodiment, a vector comprises an EF1a promoter that lacks thefirst intron of the human EF1a gene.

In a particular embodiment, it may be desirable to use a cell, celltype, cell lineage or tissue specific expression control sequence toachieve cell type specific, lineage specific, or tissue specificexpression of a desired polynucleotide sequence (e.g., to express aparticular nucleic acid encoding a polypeptide in only a subset of celltypes, cell lineages, or tissues or during specific stages ofdevelopment).

In a particular embodiment, it may be desirable to express apolynucleotide a T cell specific promoter.

As used herein, “conditional expression” may refer to any type ofconditional expression including, but not limited to, inducibleexpression; repressible expression; expression in cells or tissueshaving a particular physiological, biological, or disease state, etc.This definition is not intended to exclude cell type or tissue specificexpression. Certain embodiments provide conditional expression of apolynucleotide-of-interest, e.g., expression is controlled by subjectinga cell, tissue, organism, etc., to a treatment or condition that causesthe polynucleotide to be expressed or that causes an increase ordecrease in expression of the polynucleotide encoded by thepolynucleotide-of-interest.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc. Induceragents include, but are not limited to glucocorticoids, estrogens,mifepristone (RU486), metals, interferons, small molecules, cumate,tetracycline, doxycycline, and variants thereof.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson andKaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employedby those of skill in the art include those described in U.S. Pat. No.6,692,736. Further examples of “IRES” known in the art include, but arenot limited to IRES obtainable from picornavirus (Jackson et al., 1990)and IRES obtainable from viral or cellular mRNA sources, such as forexample, immunoglobulin heavy-chain binding protein (BiP), the vascularendothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol.18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), andinsulin-like growth factor (IGFII), the translational initiation factoreIF4G and yeast transcription factors TFIID and HAP4, theencephelomycarditis virus (EMCV) which is commercially available fromNovagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES(Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRES have also beenreported in viral genomes of Picornaviridae, Dicistroviridae andFlaviviridae species and in HCV, Friend murine leukemia virus (FrMLV)and Moloney murine leukemia virus (MoMLV).

In one embodiment, the IRES used in polynucleotides contemplated hereinis an EMCV IRES.

In particular embodiments, the polynucleotides a consensus Kozaksequence. As used herein, the term “Kozak sequence” refers to a shortnucleotide sequence that greatly facilitates the initial binding of mRNAto the small subunit of the ribosome and increases translation. Theconsensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 83), where R is apurine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987.Nucleic Acids Res. 15(20):8125-48).

Elements directing the efficient termination and polyadenylation of theheterologous nucleic acid transcripts increases heterologous geneexpression. Transcription termination signals are generally founddownstream of the polyadenylation signal. In particular embodiments,vectors comprise a polyadenylation sequence 3′ of a polynucleotideencoding a polypeptide to be expressed. The term “polyA site” or “polyAsequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript by RNApolymerase II. Polyadenylation sequences can promote mRNA stability byaddition of a polyA tail to the 3′ end of the coding sequence and thus,contribute to increased translational efficiency. Cleavage andpolyadenylation are directed by a poly(A) sequence in the RNA. The corepoly(A) sequence for mammalian pre-mRNAs has two recognition elementsflanking a cleavage-polyadenylation site. Typically, an almost invariantAAUAAA hexamer lies 20-50 nucleotides upstream of a more variableelement rich in U or GU residues. Cleavage of the nascent transcriptoccurs between these two elements and is coupled to the addition of upto 250 adenosines to the 5′ cleavage product. In particular embodiments,the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA,ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is anSV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), arabbit β-globin polyA sequence (rβgpA), variants thereof, or anothersuitable heterologous or endogenous polyA sequence known in the art. Inparticular embodiments, the poly(A) sequence is synthetic.

In particular embodiments, polynucleotides encoding one or morepolypeptides, or fusion polypeptides may be introduced into immuneeffector cells, e.g., T cells, by both non-viral and viral methods. Inparticular embodiments, delivery of one or more polynucleotides may beprovided by the same method or by different methods, and/or by the samevector or by different vectors.

The term “vector” is used herein to refer to a nucleic acid moleculecapable transferring or transporting another nucleic acid molecule. Thetransferred nucleic acid is generally linked to, e.g., inserted into,the vector nucleic acid molecule. A vector may include sequences thatdirect autonomous replication in a cell, or may include sequencessufficient to allow integration into host cell DNA. In particularembodiments, non-viral vectors are used to deliver one or morepolynucleotides contemplated herein to a T cell.

Illustrative examples of non-viral vectors include, but are not limitedto plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids,and bacterial artificial chromosomes.

Illustrative methods of non-viral delivery of polynucleotidescontemplated in particular embodiments include, but are not limited to:electroporation, sonoporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, nanoparticles, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions,DEAE-dextran-mediated transfer, gene gun, and heat-shock.

Illustrative examples of polynucleotide delivery systems suitable foruse in particular embodiments contemplated in particular embodimentsinclude, but are not limited to those provided by Amaxa Biosystems,Maxcyte, Inc., BTX Molecular Delivery Systems, and CopernicusTherapeutics Inc. Lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides have been described in the literature. See e.g., Liu etal. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal ofDrug Delivery. 2011:1-12. Antibody-targeted, bacterially derived,non-living nanocell-based delivery is also contemplated in particularembodiments.

Viral vectors comprising polynucleotides contemplated in particularembodiments can be delivered in vivo by administration to an individualpatient, typically by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application, as described below. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., mobilized peripheral blood, lymphocytes, bone marrowaspirates, tissue biopsy, etc.) or universal donor hematopoietic stemcells, followed by reimplantation of the cells into a patient.

In one embodiment, viral vectors comprising polynucleotides contemplatedherein are administered directly to an organism for transduction ofcells in vivo. Alternatively, naked DNA can be administered.Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells including, butnot limited to, injection, infusion, topical application andelectroporation. Suitable methods of administering such nucleic acidsare available and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Illustrative examples of viral vector systems suitable for use inparticular embodiments contemplated in particular embodiments include,but are not limited to, adeno-associated virus (AAV), retrovirus, herpessimplex virus, adenovirus, and vaccinia virus vectors.

In various embodiments, one or more polynucleotides encoding one or moreCD33 VHH DARIC components and/or other polypeptides contemplated hereinare introduced into an immune effector cell, e.g., T cell, bytransducing the cell with a recombinant adeno-associated virus (rAAV),comprising the one or more polynucleotides.

AAV is a small (˜26 nm) replication-defective, primarily episomal,non-enveloped virus. AAV can infect both dividing and non-dividing cellsand may incorporate its genome into that of the host cell. RecombinantAAV (rAAV) are typically composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). The ITR sequences are about 145 bp in length. In particularembodiments, the rAAV comprises ITRs and capsid sequences isolated fromAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

In some embodiments, a chimeric rAAV is used the ITR sequences areisolated from one AAV serotype and the capsid sequences are isolatedfrom a different AAV serotype. For example, a rAAV with ITR sequencesderived from AAV2 and capsid sequences derived from AAV6 is referred toas AAV2/AAV6. In particular embodiments, the rAAV vector may compriseITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment,the rAAV comprises ITR sequences derived from AAV2 and capsid sequencesderived from AAV6. In a preferred embodiment, the rAAV comprises ITRsequences derived from AAV2 and capsid sequences derived from AAV2.

In some embodiments, engineering and selection methods can be applied toAAV capsids to make them more likely to transduce cells of interest.

Construction of rAAV vectors, production, and purification thereof havebeen disclosed, e.g., in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224;8,889,641; 8,809,058; and 8,784,799, each of which is incorporated byreference herein, in its entirety.

In various embodiments, one or more polynucleotides encoding one or moreCD33 VHH DARIC components and/or other polypeptides contemplated hereinare introduced into an immune effector cell, e.g., T cell, bytransducing the cell with a retrovirus, e.g., lentivirus, comprising theone or more polynucleotides.

As used herein, the term “retrovirus” refers to an RNA virus thatreverse transcribes its genomic RNA into a linear double-stranded DNAcopy and subsequently covalently integrates its genomic DNA into a hostgenome. Illustrative retroviruses suitable for use in particularembodiments, include, but are not limited to: Moloney murine leukemiavirus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon apeleukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friendmurine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous SarcomaVirus (RSV)) and lentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include, but are notlimited to, HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred.

In various embodiments, a lentiviral vector contemplated hereincomprises one or more LTRs, and one or more, or all, of the followingaccessory elements: a cPPT/FLAP, a Psi (T) packaging signal, an exportelement, poly (A) sequences, and may optionally comprise a WPRE or HPRE,an insulator element, a selectable marker, and a cell suicide gene, asdiscussed elsewhere herein.

In particular embodiments, lentiviral vectors contemplated herein may beintegrative or non-integrating or integration defective lentivirus. Asused herein, the term “integration defective lentivirus” or “IDLV”refers to a lentivirus having an integrase that lacks the capacity tointegrate the viral genome into the genome of the host cells.Integration-incompetent viral vectors have been described in patentapplication WO 2006/010834, which is herein incorporated by reference inits entirety.

Illustrative mutations in the HIV-1 pol gene suitable to reduceintegrase activity include, but are not limited to: H12N, H12C, H16C,H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A,E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E,K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A,K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A,Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A,R262A, R263A and K264H.

The term “long terminal repeat (LTR)” refers to domains of base pairslocated at the ends of retroviral DNAs which, in their natural sequencecontext, are direct repeats and contain U3, R and U5 regions.

As used herein, the term “FLAP element” or “cPPT/FLAP” refers to anucleic acid whose sequence includes the central polypurine tract andcentral termination sequences (cPPT and CTS) of a retrovirus, e.g.,HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No.6,682,907 and in Zennou, et al., 2000, Cell, 101:173.

As used herein, the term “packaging signal” or “packaging sequence”refers to psi [Ψ] sequences located within the retroviral genome whichare required for insertion of the viral RNA into the viral capsid orparticle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4;pp. 2101-2109.

The term “export element” refers to a cis-acting post-transcriptionalregulatory element which regulates the transport of an RNA transcriptfrom the nucleus to the cytoplasm of a cell. Examples of RNA exportelements include, but are not limited to, the human immunodeficiencyvirus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991.J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and thehepatitis B virus post-transcriptional regulatory element (HPRE).

In particular embodiments, expression of heterologous sequences in viralvectors is increased by incorporating posttranscriptional regulatoryelements, efficient polyadenylation sites, and optionally, transcriptiontermination signals into the vectors. A variety of posttranscriptionalregulatory elements can increase expression of a heterologous nucleicacid at the protein, e.g., woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886);the posttranscriptional regulatory element present in hepatitis B virus(HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu etal., 1995, Genes Dev., 9:1766).

Lentiviral vectors preferably contain several safety enhancements as aresult of modifying the LTRs. “Self-inactivating” (SIN) vectors refersto replication-defective vectors, e.g., retroviral or lentiviralvectors, in which the right (3′) LTR enhancer-promoter region, known asthe U3 region, has been modified (e.g., by deletion or substitution) toprevent viral transcription beyond the first round of viral replication.Self-inactivation is preferably achieved through in the introduction ofa deletion in the U3 region of the 3′ LTR of the vector DNA, i.e., theDNA used to produce the vector RNA. Thus, during reverse transcription,this deletion is transferred to the 5′ LTR of the proviral DNA. Inparticular embodiments, it is desirable to eliminate enough of the U3sequence to greatly diminish or abolish altogether the transcriptionalactivity of the LTR, thereby greatly diminishing or abolishing theproduction of full-length vector RNA in transduced cells. In the case ofHIV based lentivectors, it has been discovered that such vectorstolerate significant U3 deletions, including the removal of the LTR TATAbox (e.g., deletions from −418 to −18), without significant reductionsin vector titers.

An additional safety enhancement is provided by replacing the U3 regionof the 5′ LTR with a heterologous promoter to drive transcription of theviral genome during production of viral particles. Examples ofheterologous promoters which can be used include, for example, viralsimian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV)(e.g., immediate early), Moloney murine leukemia virus (MoMLV), Roussarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase)promoters.

The terms “pseudotype” or “pseudotyping” as used herein, refer to avirus whose viral envelope proteins have been substituted with those ofanother virus possessing preferable characteristics. For example, HIVcan be pseudotyped with vesicular stomatitis virus G-protein (VSV-G)envelope proteins, which allows HIV to infect a wider range of cellsbecause HIV envelope proteins (encoded by the env gene) normally targetthe virus to CD4⁺ presenting cells.

In certain embodiments, lentiviral vectors are produced according toknown methods. See e.g., Kutner et al., BMC Biotechnol. 2009; 9:10. doi:10.1186/1472-6750-9-10; Kutner et al. Nat. Protoc. 2009; 4(4):495-505.doi: 10.1038/nprot.2009.22.

According to certain specific embodiments contemplated herein, most orall of the viral vector backbone sequences are derived from alentivirus, e.g., HIV-1. However, it is to be understood that manydifferent sources of retroviral and/or lentiviral sequences can be used,or combined and numerous substitutions and alterations in certain of thelentiviral sequences may be accommodated without impairing the abilityof a transfer vector to perform the functions described herein.Moreover, a variety of lentiviral vectors are known in the art, seeNaldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dullet al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which maybe adapted to produce a viral vector or transfer plasmid contemplatedherein.

In various embodiments, one or more polynucleotides encoding one or moreCD33 VHH DARIC components and/or other polypeptides contemplated hereinare introduced into an immune effector cell, by transducing the cellwith an adenovirus comprising the one or more polynucleotides.

Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and high levels of expression have beenobtained. This vector can be produced in large quantities in arelatively simple system. Most adenovirus vectors are engineered suchthat a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequentlythe replication defective vector is propagated in human 293 cells thatsupply deleted gene function in trans. Ad vectors can transduce multipletypes of tissues in vivo, including non-dividing, differentiated cellssuch as those found in liver, kidney and muscle. Conventional Ad vectorshave a large carrying capacity.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, may utilize a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones & Shenk, 1978), the current adenovirus vectors, with the help of293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham & Prevec, 1991). Adenovirus vectors have been used in eukaryoticgene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) andvaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).Studies in administering recombinant adenovirus to different tissuesinclude trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al.,1992), muscle injection (Ragot et al., 1993), peripheral intravenousinjections (Herz & Gerard, 1993) and stereotactic inoculation into thebrain (Le Gal La Salle et al., 1993). An example of the use of an Advector in a clinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)).

In various embodiments, one or more polynucleotides encoding one or moreCD33 VHH DARIC components and/or other polypeptides contemplated hereinare introduced into an immune effector cell by transducing the cell witha herpes simplex virus, e.g., HSV-1, HSV-2, comprising the one or morepolynucleotides.

The mature HSV virion consists of an enveloped icosahedral capsid with aviral genome consisting of a linear double-stranded DNA molecule that is152 kb. In one embodiment, the HSV based viral vector is deficient inone or more essential or non-essential HSV genes. In one embodiment, theHSV based viral vector is replication deficient. Most replicationdeficient HSV vectors contain a deletion to remove one or moreintermediate-early, early, or late HSV genes to prevent replication. Forexample, the HSV vector may be deficient in an immediate early geneselected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and acombination thereof. Advantages of the HSV vector are its ability toenter a latent stage that can result in long-term DNA expression and itslarge viral DNA genome that can accommodate exogenous DNA inserts of upto 25 kb. HSV-based vectors are described in, for example, U.S. Pat.Nos. 5,837,532, 5,846,782, and 5,804,413, and International PatentApplications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583,each of which are incorporated by reference herein in its entirety.

G. Genetically Modified Cells

In various embodiments, cells are modified to express a CD33 VHH DARIC,one or more CD33 VHH DARIC components, an anti-CD33 VHH CAR, and/orfusion proteins contemplated herein, for use in the treatment of cancer.Cells may be non-genetically modified to express one or more of thepolypeptides contemplated herein, or in particular preferredembodiments, cells may be genetically modified to express one or more ofthe polypeptides contemplated herein. As used herein, the term“genetically engineered” or “genetically modified” refers to theaddition of extra genetic material in the form of DNA or RNA into thetotal genetic material in a cell. The terms, “genetically modifiedcells,” “modified cells,” and “redirected cells,” are usedinterchangeably in particular embodiments.

In particular embodiments, one or more CD33 VHH DARIC components or ananti-CD33 VHH CAR contemplated herein are introduced and expressed inimmune effector cells to improve the efficacy of the immune effectorcells.

An “immune effector cell,” is any cell of the immune system that has oneor more effector functions (e.g., cytotoxic cell killing activity,secretion of cytokines, induction of ADCC and/or CDC). The illustrativeimmune effector cells contemplated herein are T lymphocytes, includingbut not limited to cytotoxic T cells (CTLs; CD8⁺ T cells), TILs, andhelper T cells (HTLs; CD4⁺ T cells). In a particular embodiment, thecells comprise αβ T cells. In a particular embodiment, the cellscomprise γδ T cells. In one embodiment, immune effector cells includenatural killer (NK) cells. In one embodiment, immune effector cellsinclude natural killer T (NKT) cells. Immune effector cells can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic).

“Autologous,” as used herein, refers to cells from the same subject.“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison. “Syngeneic,” as usedherein, refers to cells of a different subject that are geneticallyidentical to the cell in comparison. “Xenogeneic,” as used herein,refers to cells of a different species to the cell in comparison. Inpreferred embodiments, the cells are human autologous immune effectorcells.

Illustrative immune effector cells suitable for introducing one or moreCD33 VHH DARIC components or an anti-CD33 VHH CAR contemplated hereininclude T lymphocytes. The terms “T cell” or “T lymphocyte” areart-recognized and are intended to include thymocytes, immature Tlymphocytes, mature T lymphocytes, resting T lymphocytes, or activated Tlymphocytes. A T cell can be a T helper (Th) cell, for example a Thelper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a helper Tcell (HTL; CD4⁺ T cell), a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+T cell, CD4⁻CD8⁻ T cell, or any other subset of T cells. In particularembodiments, the T cell expresses a T cell receptor. T cell receptorscomprise two subunits, an alpha chain and a beta chain subunit (αβTCR),or a gamma chain and a delta chain subunit (γδTCR), each of which is aunique protein produced by recombination event in each T cell's genome.In particular embodiments, a T cell is an αβTCR T cell (an αβ T cell).In particular embodiments, a T cell is a γδTCR T cell (a γδ T cell).Other illustrative populations of T cells suitable for use in particularembodiments include naïve T cells and memory T cells.

As would be understood by the skilled person, other cells may also beused as immune effector cells comprising one or more CD33 VHH DARICcomponents or an anti-CD33 VHH CAR contemplated herein. In particularembodiments, immune effector cells also include NK cells, NKT cells,neutrophils, and macrophages. Immune effector cells also includeprogenitors of effector cells wherein such progenitor cells can beinduced to differentiate into immune effector cells in vivo or in vitro.Thus, in particular embodiments, immune effector cells includeprogenitors of immune effectors cells such as hematopoietic stem cells(HSCs) contained within the CD34⁺ population of cells derived from cordblood, bone marrow or mobilized peripheral blood which uponadministration in a subject differentiate into mature immune effectorcells, or which can be induced in vitro to differentiate into matureimmune effector cells.

The term, “CD34⁺ cell,” as used herein refers to a cell expressing theCD34 protein on its cell surface. “CD34,” as used herein refers to acell surface glycoprotein (e.g., sialomucin protein) that often acts asa cell-cell adhesion factor and is involved in T cell entrance intolymph nodes. The CD34⁺ cell population contains hematopoietic stem cells(HSC), which upon administration to a patient differentiate andcontribute to all hematopoietic lineages, including T cells, NK cells,NKT cells, neutrophils and cells of the monocyte/macrophage lineage.

Methods for making the immune effector cells which express one or moreCD33 VHH DARIC components or an anti-CD33 VHH CAR contemplated hereinare provided in particular embodiments. In one embodiment, the methodcomprises transfecting or transducing immune effector cells isolatedfrom an individual such that the immune effector cells with one or morenucleic acids and/or vectors, e.g., a lentiviral vector comprising anucleic acid encoding one or more CD33 VHH DARIC components or ananti-CD33 VHH CAR contemplated herein. In one embodiment, the methodcomprises transfecting or transducing immune effector cells isolatedfrom an individual such that the immune effector cells express one ormore CD33 VHH DARIC components or an anti-CD33 VHH CAR contemplatedherein. In certain embodiments, the immune effector cells are isolatedfrom an individual and genetically modified without further manipulationin vitro. Such cells can then be directly re-administered into theindividual. In further embodiments, the immune effector cells are firstactivated and stimulated to proliferate in vitro prior to beinggenetically modified. In this regard, the immune effector cells may becultured before and/or after being genetically modified.

In particular embodiments, prior to in vitro manipulation or geneticmodification of the immune effector cells described herein, the sourceof cells is obtained from a subject. In particular embodiments, themodified immune effector cells comprise T cells.

T cells can be obtained from a number of sources including, but notlimited to, peripheral blood mononuclear cells, bone marrow, lymph nodestissue, cord blood, thymus issue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled person,such as sedimentation, e.g., FICOLL™ separation.

In other embodiments, an isolated or purified population of T cells isused. In some embodiments, after isolation of PBMC, both cytotoxic andhelper T lymphocytes can be sorted into naïve, memory, and effector Tcell subpopulations either before or after activation, expansion, and/orgenetic modification.

In one embodiment, an isolated or purified population of T cellsexpresses one or more of the markers including, but not limited to aCD3⁺, CD4⁺, CD8⁺, or a combination thereof.

In certain embodiments, the T cells are isolated from an individual andfirst activated and stimulated to proliferate in vitro prior to beingmodified to express one or more CD33 VHH DARIC components or ananti-CD33 VHH CAR.

In order to achieve sufficient therapeutic doses of T cell compositions,T cells are often subjected to one or more rounds of stimulation,activation and/or expansion. In particular embodiments, T cells can beactivated and expanded generally using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041,each of which is incorporated herein by reference in its entirety. Inparticular embodiments, T cells are activated and expanded for about 6hours, about 12 hours, about 18 hours or about 24 hours prior tointroduction of vectors or polynucleotides encoding one or more CD33 VHHDARIC components or an anti-CD33 VHH CAR contemplated herein.

H. Compositions and Formulations

The compositions contemplated herein may comprise one or more CD33 VHHDARIC components or an anti-CD33 VHH CAR, polynucleotides encoding oneor more CD33 VHH DARIC components or an anti-CD33 VHH CAR, vectorscomprising same, genetically modified immune effector cells, bridgingfactors, etc. Compositions include, but are not limited to,pharmaceutical compositions. A “pharmaceutical composition” refers to acomposition formulated in pharmaceutically-acceptable orphysiologically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy. It will also be understood that, if desired, thecompositions may be administered in combination with other agents aswell, such as, e.g., cytokines, growth factors, hormones, smallmolecules, chemotherapeutics, pro-drugs, drugs, antibodies, or othervarious pharmaceutically-active agents. There is virtually no limit toother components that may also be included in the compositions, providedthat the additional agents do not adversely affect the ability of thecomposition to deliver the intended therapy.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “pharmaceutically acceptable carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the bridging factors,polypeptides, polynucleotides, vectors comprising same, or geneticallymodified immune effector cells are administered. Illustrative examplesof pharmaceutical carriers can be sterile liquids, such as cell culturemedia, water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients inparticular embodiments, include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

In one embodiment, a composition comprising a pharmaceuticallyacceptable carrier is suitable for administration to a subject. Inparticular embodiments, a composition comprising a carrier is suitablefor parenteral administration, e.g., intravascular (intravenous orintraarterial), intraperitoneal or intramuscular administration. Inparticular embodiments, a composition comprising a pharmaceuticallyacceptable carrier is suitable for intraventricular, intraspinal, orintrathecal administration. Pharmaceutically acceptable carriers includesterile aqueous solutions, cell culture media, or dispersions. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the bridging factors, polypeptides, polynucleotides,vectors comprising same, or genetically modified immune effector cells,use thereof in the pharmaceutical compositions is contemplated.

In particular embodiments, compositions contemplated herein comprisegenetically modified T cells and a pharmaceutically acceptable carrier.A composition comprising a cell-based composition contemplated hereincan be administered separately by enteral or parenteral administrationmethods or in combination with other suitable compounds to effect thedesired treatment goals.

In particular embodiments, compositions contemplated herein comprise abridging factor and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier must be of sufficiently highpurity and of sufficiently low toxicity to render it suitable foradministration to the human subject being treated. It further shouldmaintain or increase the stability of the composition. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with othercomponents of the composition. For example, the pharmaceuticallyacceptable carrier can be, without limitation, a binding agent (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.), a filler (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant(e.g., magnesium stearate, talc, silica, colloidal silicon dioxide,stearic acid, metallic stearates, hydrogenated vegetable oils, cornstarch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), adisintegrant (e.g., starch, sodium starch glycolate, etc.), or a wettingagent (e.g., sodium lauryl sulfate, etc.). Other suitablepharmaceutically acceptable carriers for the compositions contemplatedherein include, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs,silicic acids, viscous paraffins, hydroxymethylcelluloses,polyvinylpyrrolidones and the like.

Such carrier solutions also can contain buffers, diluents and othersuitable additives. The term “buffer” as used herein refers to asolution or liquid whose chemical makeup neutralizes acids or baseswithout a significant change in pH. Examples of buffers contemplatedherein include, but are not limited to, Dulbecco's phosphate bufferedsaline (PBS), Ringer's solution, 5% dextrose in water (D5W),normal/physiologic saline (0.9% NaCl).

The pharmaceutically acceptable carriers may be present in amountssufficient to maintain a pH of the composition of about 7.Alternatively, the composition has a pH in a range from about 6.8 toabout 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still anotherembodiment, the composition has a pH of about 7.4.

Compositions contemplated herein may comprise a nontoxicpharmaceutically acceptable medium. The compositions may be asuspension. The term “suspension” as used herein refers to non-adherentconditions in which cells are not attached to a solid support. Forexample, cells maintained as a suspension may be stirred or agitated andare not adhered to a support, such as a culture dish.

In particular embodiments, compositions contemplated herein areformulated in a suspension, where the modified T cells are dispersedwithin an acceptable liquid medium or solution, e.g., saline orserum-free medium, in an intravenous (IV) bag or the like. Acceptablediluents include, but are not limited to water, PlasmaLyte, Ringer'ssolution, isotonic sodium chloride (saline) solution, serum-free cellculture medium, and medium suitable for cryogenic storage, e.g.,Cryostor® medium.

In certain embodiments, a pharmaceutically acceptable carrier issubstantially free of natural proteins of human or animal origin, andsuitable for storing a composition comprising a population of modified Tcells. The therapeutic composition is intended to be administered into ahuman patient, and thus is substantially free of cell culture componentssuch as bovine serum albumin, horse serum, and fetal bovine serum.

In some embodiments, compositions are formulated in a pharmaceuticallyacceptable cell culture medium. Such compositions are suitable foradministration to human subjects. In particular embodiments, thepharmaceutically acceptable cell culture medium is a serum free medium.

Serum-free medium has several advantages over serum containing medium,including a simplified and better-defined composition, a reduced degreeof contaminants, elimination of a potential source of infectious agents,and lower cost. In various embodiments, the serum-free medium isanimal-free, and may optionally be protein-free. Optionally, the mediummay contain biopharmaceutically acceptable recombinant proteins.“Animal-free” medium refers to medium wherein the components are derivedfrom non-animal sources. Recombinant proteins replace native animalproteins in animal-free medium and the nutrients are obtained fromsynthetic, plant or microbial sources. “Protein-free” medium, incontrast, is defined as substantially free of protein.

Illustrative examples of serum-free media used in particularcompositions includes, but is not limited to, QBSF-60 (QualityBiological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

In one embodiment, the compositions comprising modified T cells areformulated in PlasmaLyte.

In various embodiments, compositions comprising modified T cells areformulated in a cryopreservation medium. For example, cryopreservationmedia with cryopreservation agents may be used to maintain a high cellviability outcome post-thaw. Illustrative examples of cryopreservationmedia used in particular compositions includes, but is not limited to,CryoStor CS10, CryoStor CSS, and CryoStor CS2.

In one embodiment, the compositions are formulated in a solutioncomprising 50:50 PlasmaLyte A to CryoStor CS10.

In particular embodiments, the composition is substantially free ofmycoplasma, endotoxin, and microbial contamination. By “substantiallyfree” with respect to endotoxin is meant that there is less endotoxinper dose of cells than is allowed by the FDA for a biologic, which is atotal endotoxin of 5 EU/kg body weight per day, which for an average 70kg person is 350 EU per total dose of cells. In particular embodiments,compositions contemplated herein contain about 0.5 EU/ml to about 5.0EU/ml, or about 0.5 EU/ml, 1.0 EU/ml, 1.5 EU/ml, 2.0 EU/ml, 2.5 EU/ml,3.0 EU/ml, 3.5 EU/ml, 4.0 EU/ml, 4.5 EU/ml, or 5.0 EU/ml.

In particular embodiments, formulation of pharmaceutically-acceptablecarrier solutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., enteral and parenteral, e.g., intravascular,intravenous, intrarterial, intraosseously, intraventricular,intracerebral, intracranial, intraspinal, intrathecal, andintramedullary administration and formulation. It would be understood bythe skilled artisan that particular embodiments contemplated herein maycomprise other formulations, such as those that are well known in thepharmaceutical art, and are described, for example, in Remington: TheScience and Practice of Pharmacy, volume I and volume II. 22^(nd)Edition. Edited by Loyd V. Allen Jr. Philadelphia, Pa.: PharmaceuticalPress; 2012, which is incorporated by reference herein, in its entirety.

In particular embodiments, compositions comprise an amount of immuneeffector cells comprising a polynucleotide encoding one or more CD33 VHHDARIC components or an anti-CD33 VHH CAR contemplated herein. Inparticular embodiments, compositions comprise an amount of immuneeffector cells that express one or more CD33 VHH DARIC components or ananti-CD33 VHH CAR contemplated herein. As used herein, the term “amount”refers to “an amount effective” or “an effective amount” of cellscomprising one or more CD33 VHH DARIC components or an anti-CD33 VHH CARcontemplated herein, etc., to achieve a beneficial or desiredprophylactic or therapeutic result in the presence of a bridging factor,including clinical results.

A “prophylactically effective amount” refers to an amount of cellscomprising one or more CD33 VHH DARIC components or an anti-CD33 VHH CARcontemplated herein, etc., effective to achieve the desired prophylacticresult in the presence of a bridging factor. Typically but notnecessarily, since a prophylactic dose is used in subjects prior to orat an earlier stage of disease, the prophylactically effective amount isless than the therapeutically effective amount.

A “therapeutically effective amount” refers to an amount of cellscomprising one or more CD33 VHH DARIC components or an anti-CD33 VHH CARcontemplated herein that is effective to “treat” a subject (e.g., apatient) in the presence of a bridging factor. When a therapeutic amountis indicated, the precise amount of the compositions to be administered,cells, bridging factor, etc, can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject).

It can generally be stated that a pharmaceutical composition comprisingthe immune effector cells described herein may be administered at adosage of 10² to 10¹⁰ cells/kg body weight, preferably 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.The number of cells will depend upon the ultimate use for which thecomposition is intended as will the type of cells included therein. Foruses provided herein, the cells are generally in a volume of a liter orless, can be 500 mls or less, even 250 mls or 100 mls or less. Hence thedensity of the desired cells is typically greater than 10⁶ cells/ml andgenerally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml orgreater. The clinically relevant number of immune cells can beapportioned into multiple, infusions that cumulatively equal or exceed10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹² cells. In some embodiments,particularly since all the infused cells will be redirected to aparticular target antigen, lower numbers of cells, in the range of10⁶/kilogram (10⁶-10¹¹ per patient) may be administered.

If desired, the treatment may also include administration of mitogens(e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN-γ,IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13,Flt3-L, RANTES, MIPla, etc.) as described herein to enhance induction ofthe immune response.

Generally, compositions comprising the cells activated and expanded asdescribed herein may be utilized in the treatment and prevention ofdiseases that arise in individuals who are immunocompromised. Inparticular, compositions contemplated herein are used in the treatmentof cancer. In particular embodiments, the immune effector cells may beadministered either alone, or as a pharmaceutical composition incombination with carriers, diluents, excipients, and/or with othercomponents such as IL-2 or other cytokines or cell populations.

In particular embodiments, pharmaceutical compositions comprise anamount of genetically modified T cells, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients.

In particular embodiments, pharmaceutical compositions comprise anamount of bridging factor, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients.

In a particular embodiment, compositions comprise an effective amount ofimmune effector cells comprising one or more CD33 VHH DARIC componentsor an anti-CD33 VHH CAR contemplated herein, alone or in combinationwith a bridging factor and/or one or more therapeutic agents, such asradiation therapy, chemotherapy, transplantation, immunotherapy, hormonetherapy, photodynamic therapy, etc. The compositions may also beadministered in combination with antibiotics. Such therapeutic agentsmay be accepted in the art as a standard treatment for a particulardisease state as described herein, such as a particular cancer.Exemplary therapeutic agents contemplated include cytokines, growthfactors, steroids, NSAIDs, DMARDs, anti-inflammatories,chemotherapeutics, radiotherapeutics, therapeutic antibodies, or otheractive and ancillary agents.

In a particular embodiment, a composition comprising an effective amountof immune effector cells comprising a polynucleotide encoding one ormore CD33 VHH DARIC components or an anti-CD33 VHH CAR contemplatedherein is administered to a subject, and a composition comprising aneffective amount of a bridging factor is administered to the subject,before, during, in combination with or subsequently to the cellularcomposition, and optionally repetitively administered to the subject.

In certain embodiments, compositions comprising immune effector cellscomprising a polynucleotide encoding one or more CD33 VHH DARICcomponents or an anti-CD33 VHH CAR contemplated herein may beadministered in conjunction with any number of anti-inflammatory agents,chemotherapeutic agents, or therapeutic antibodies, and the like.

I. Therapeutic Methods

Immune effector cells modified to express a polynucleotide encoding oneor more CD33 VHH DARIC components or an anti-CD33 VHH CAR contemplatedherein provide improved methods of adoptive immunotherapy for use in theprevention, treatment, and amelioration of, or for preventing, treating,or ameliorating at least one symptom associated with an immune disorder,e.g., cancer.

Immune effector cells comprising a CD33 DARIC signaling component, aCD33 VHH DARIC binding component, or an anti-CD33 VHH CAR provideimproved methods of adoptive immunotherapy for use in the prevention,treatment, and amelioration of, or for preventing, treating, orameliorating at least one symptom associated with an immune disorder,e.g., cancer.

In particular embodiments, immune effector cells modified to express aCD33 VHH DARIC provide improved methods of adoptive immunotherapy tofine-tune the safety and efficacy of a cytotoxic response against targetcells, e.g., tumor cells, expressing target antigens while decreasingthe risk of on-target antigen, off-target cell cytotoxicity (recognizingthe target antigen on a normal, non-target cell).

In particular embodiments, a method of preventing, treating, orameliorating at least one symptom of a cancer comprises administeringthe subject an effective amount of modified immune effector cells or Tcells comprising one or more components of a CD33 VHH DARIC or ananti-CD33 VHH CAR to redirect the cells to a target cell. Thegenetically modified cells are a more efficacious and safe cellularimmunotherapy by virtue of transducing a chemically regulatableimmunostimulatory signal.

In particular embodiments, one or more immune effector cells, e.g., Tcells, are modified to express both a CD33 VHH DARIC binding componentand a CD33 DARIC signaling component. In this case, the modified cellsare administered to a subject in need thereof and home to the targetcells via the interaction of the CD33 VHH binding component expressed onthe immune effector cell and CD33 expressed on the target cell. Abridging factor is administered to the subject before the modifiedcells, about the same time as the modified cells, or after the modifiedcells have been administered to the subject. In the presence of thebridging factor, a ternary complex forms between the CD33 VHH DARICbinding component, the bridging factor, and the CD33 DARIC signalingcomponent. Upon formation of the ternary complex, the CD33 VHH DARICtransduces an immunostimulatory signal to the immune effector cell thatin turn, elicits a cytotoxic response from the immune effector cellagainst the target cell.

In particular embodiments, one or more immune effector cells, e.g., Tcells, are modified to express a CD33 DARIC signaling component. In thiscase, the modified cells are administered to a subject in need thereof.A CD33 VHH DARIC binding component can be administered to the subjectbefore the modified cells, about the same time as the modified cells, orafter the modified cells have been administered to the subject. Inaddition, the CD33 VHH DARIC binding component can be administered tothe subject in a preformed complex with the bridging factor; at the sametime as the bridging factor, but in a separate composition; or at adifferent time than the bridging factor. The CD33 VHH binding componentbinds CD33 expressed on the target cell, either in the presence orabsence of the bridging factor. In the presence of the bridging factor,a ternary complex forms between the CD33 VHH DARIC binding component,the bridging factor, and the CD33 DARIC signaling component. Uponformation of the ternary complex, the CD33 VHH DARIC transduces animmunostimulatory signal to the immune effector cell that in turn,elicits a cytotoxic response from the immune effector cell against thetarget cell.

In particular embodiments, one or more immune effector cells, e.g., Tcells, are modified to express the CD33 DARIC signaling component. Inthis case, the modified cells are administered to a subject in needthereof. A CD33 VHH DARIC binding component can be administered to thesubject before the modified cells, about the same time as the modifiedcells, or after the modified cells have been administered to thesubject. In addition, the CD33 VHH DARIC binding component can beadministered to the subject in a preformed complex with the bridgingfactor; at the same time as the bridging factor, but in a separatecomposition; or at a different time than the bridging factor. The CD33binding component binds the target antigen expressed on the target cell,either in the presence or absence of the bridging factor. In thepresence of the bridging factor, a ternary complex forms between theCD33 VHH DARIC binding component, the bridging factor, and the CD33DARIC signaling component. Upon formation of the ternary complex, theCD33 VHH DARIC transduces an immunostimulatory signal to the immuneeffector cell that in turn, elicits a cytotoxic response from the immuneeffector cell against the target cell. In particular embodiments, CD33VHH DARIC activation can be induced in cases where remission orregression is incomplete and the condition relapses or becomesrefractory to treatment.

In particular preferred embodiments, the specificity of a primary T cellis redirected to tumor or cancer cells that express CD33 by geneticallymodifying a T cell, e.g., a primary T cell, with one or more CD33 VHHDARIC components.

In particular preferred embodiments, the specificity of a primary T cellis redirected to tumor or cancer cells that express CD33 by geneticallymodifying a T cell, e.g., a primary T cell, with an engineered antigenreceptor directed to the target antigen and one or more CD33 VHH DARICcomponents.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors orcancers.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors or cancersincluding, but not limited to: adrenal cancer, adrenocortical carcinoma,anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, bladder cancer, bonecancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiactumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma,colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma insitu (DCIS) endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibroushistiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumor(GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer,hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer,intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer,leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer,non-small cell lung cancer, lung carcinoid tumor, malignantmesothelioma, medullary carcinoma, medulloblastoma, menangioma,melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer,myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms,nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,pancreatic islet cell tumors, papillary carcinoma, paraganglioma,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritonealcancer, prostate cancer, rectal cancer, retinoblastoma, renal cellcarcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivarygland cancer, sebaceous gland carcinoma, skin cancer, soft tissuesarcoma, squamous cell carcinoma, small cell lung cancer, smallintestine cancer, stomach cancer, sweat gland carcinoma, synovioma,testicular cancer, throat cancer, thymus cancer, thyroid cancer,urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer,vascular cancer, vulvar cancer, and Wilms Tumor.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors or cancersincluding, without limitation, non-small cell lung carcinoma, head andneck squamous cell carcinoma, colorectal cancer, pancreatic cancer,breast cancer, thyroid cancer, bladder cancer, cervical cancer,esophageal cancer, ovarian cancer, gastric cancer endometrial cancer,gliomas, glioblastomas, and oligodendroglioma.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors or cancersincluding, without limitation, non-small-cell lung cancer, metastaticcolorectal cancer, glioblastoma, head and neck cancer, pancreaticcancer, and breast cancer.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of glioblastoma.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of liquid cancers orhematological cancers.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of B-cell malignancies,including but not limited to: leukemias, lymphomas, and multiplemyeloma.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of liquid cancersincluding, but not limited to leukemias, lymphomas, and multiplemyelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia(CLL), and chronic myeloid leukemia (CIVIL), chronic myelomonocyticleukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodularlymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, smalllymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicularlymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosisfungoides, anaplastic large cell lymphoma, Sézary syndrome, precursorT-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma,smoldering multiple myeloma, plasma cell leukemia, non-secretorymyeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma ofbone, and extramedullary plasmacytoma.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of acute myeloid leukemia(AML).

Preferred cells for use in the methods contemplated herein includeautologous/autogeneic (“self”) cells, preferably hematopoietic cells,more preferably T cells, and more preferably immune effector cells.

In particular embodiments, a method comprises administering atherapeutically effective amount of modified immune effector cells thatexpress one or more CD33 VHH DARIC components, to a patient in needthereof, and also administering a bridging factor to the subject. Incertain embodiments, the cells are used in the treatment of patients atrisk for developing an immune disorder. Thus, particular embodimentscomprise the treatment or prevention or amelioration of at least onesymptom of an immune disorder, e.g., cancer, comprising administering toa subject in need thereof, a therapeutically effective amount of themodified immune effector cells contemplated herein and a bridgingfactor.

In particular embodiments, a method comprises administering atherapeutically effective amount of modified immune effector cells thatexpress an anti-CD33 VHH CAR to a patient in need thereof. In certainembodiments, the cells are used in the treatment of patients at risk fordeveloping an immune disorder. Thus, particular embodiments comprise thetreatment or prevention or amelioration of at least one symptom of animmune disorder, e.g., cancer, comprising administering to a subject inneed thereof, a therapeutically effective amount of the modified immuneeffector cells contemplated herein and a bridging factor.

In particular embodiments, a method comprises administering atherapeutically effective amount of modified immune effector cells thatexpress a CD33 DARIC signaling component or a composition comprising thesame, to a patient in need thereof, and also administering a CD33 VHHDARIC binding component and a bridging factor, optionally wherein theCD33 VHH DARIC binding component is bound to the bridging factor priorto administration, to the subject. In certain embodiments, the cells areused in the treatment of patients at risk for developing an immunedisorder. Thus, particular embodiments comprise the treatment orprevention or amelioration of at least one symptom of an immunedisorder, e.g., cancer, comprising administering to a subject in needthereof, a therapeutically effective amount of the modified immuneeffector cells that express a CD33 DARIC signaling component andoptionally and engineered antigen receptor or another DARIC bindingcomponent, a CD33 VHH DARIC binding component, and a bridging factor.

The quantity and frequency of administration of modified immune effectorcells, CD33 DARIC VHH binding components, and/or bridging factor will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages anddose schedules may be determined by clinical trials.

In one illustrative embodiment, the effective amount of modified immuneeffector cells provided to a subject is at least 2×10⁶ cells/kg, atleast 3×10⁶ cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg,at least 6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶cells/kg, at least 9×10⁶ cells/kg, or at least 10×10⁶ cells/kg, or morecells/kg, including all intervening doses of cells.

In another illustrative embodiment, the effective amount of modifiedimmune effector cells provided to a subject is about 2×10⁶ cells/kg,about 3×10⁶ cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about6×10⁶ cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, or about 10×10⁶ cells/kg, or more cells/kg, including allintervening doses of cells.

In another illustrative embodiment, the effective amount of modifiedimmune effector cells provided to a subject is from about 2×10⁶ cells/kgto about 10×10⁶ cells/kg, about 3×10⁶ cells/kg to about 10×10⁶ cells/kg,about 4×10⁶ cells/kg to about 10×10⁶ cells/kg, about 5×10⁶ cells/kg toabout 10×10⁶ cells/kg, 2×10⁶ cells/kg to about 6×10⁶ cells/kg, 2×10⁶cells/kg to about 7×10⁶ cells/kg, 2×10⁶ cells/kg to about 8×10⁶cells/kg, 3×10⁶ cells/kg to about 6×10⁶ cells/kg, 3×10⁶ cells/kg toabout 7×10⁶ cells/kg, 3×10⁶ cells/kg to about 8×10⁶ cells/kg, 4×10⁶cells/kg to about 6×10⁶ cells/kg, 4×10⁶ cells/kg to about 7×10⁶cells/kg, 4×10⁶ cells/kg to about 8×10⁶ cells/kg, 5×10⁶ cells/kg toabout 6×10⁶ cells/kg, 5×10⁶ cells/kg to about 7×10⁶ cells/kg, 5×10⁶cells/kg to about 8×10⁶ cells/kg, or 6×10⁶ cells/kg to about 8×10⁶cells/kg, including all intervening doses of cells.

One of ordinary skill in the art would recognize that multipleadministrations of the compositions contemplated in particularembodiments may be required to effect the desired therapy. For example,a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10years, or more. Modified immune effector cells, CD33 VHH DARICcomponents, and bridging factor may be administered in the same ordifferent compositions; in one or more compositions at the same time; ormore than one composition at different times. Modified immune effectorcells, CD33 VHH DARIC components, and bridging factor may beadministered through the same route of administration or differentroutes.

In certain embodiments, it may be desirable to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom, and reinfuse thepatient with these activated and expanded T cells. This process can becarried out multiple times every few weeks. In certain embodiments, Tcells can be activated from blood draws of from 10 cc to 400 cc. Incertain embodiments, T cells are activated from blood draws of 20 cc, 30cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc,250 cc, 300 cc, 350 cc, or 400 cc or more. Not to be bound by theory,using this multiple blood draw/multiple reinfusion protocol may serve toselect out certain populations of T cells.

In one embodiment, a method of treating a subject diagnosed with acancer, comprises removing immune effector cells from the subject,modifying the immune effector cells by introducing one or more vectorsencoding one or more CD33 VHH DARIC components into the cell andproducing a population of modified immune effector cells, andadministering the population of modified immune effector cells to thesame subject. In a preferred embodiment, the immune effector cellscomprise T cells.

In one embodiment, a method of treating a subject diagnosed with acancer, comprises removing immune effector cells from the subject,modifying the immune effector cells by introducing one or more vectorsencoding an anti-CD33 VHH CAR into the cell and producing a populationof modified immune effector cells, and administering the population ofmodified immune effector cells to the same subject. In a preferredembodiment, the immune effector cells comprise T cells.

The methods for administering the cell compositions contemplated inparticular embodiments include any method which is effective to resultin reintroduction of ex vivo modified immune effector cells orreintroduction of modified progenitors of immune effector cells thatupon introduction into a subject differentiate into mature immuneeffector cells. One method comprises modifying peripheral blood T cellsex vivo by introducing one or more vectors encoding one or more CD33 VHHDARIC components or an anti-CD33 VHH CAR into the cell and returning thetransduced cells into the subject.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings contemplated herein that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified in particular embodimentsto yield essentially similar results.

EXAMPLES Example 1 CD33 VHH DARIC T Cells Exhibit Anti-Tumor Responses

Anti-CD33 VHH DARIC binding and signaling components were designed,constructed, and verified. CD33 specific VHH DARIC lentiviral vectorswere constructed comprising an MNDU3 promoter operably linked to apolynucleotide encoding: a DARIC signaling component (CD8α-signalpeptide, an FRB variant (T82L), a CD8a transmembrane domain, anintracellular 4-1BB costimulatory domain, and a CD3 zeta signalingdomain); a P2A sequence; and a DARIC binding component (an Igκ-signalpeptide, a CD33 specific VHH binding domain (camelid or humanized), aG45 linker, an FKBP12 domain, and a CD4 derived transmembrane domainwith a truncated intracellular domain (FIG. 1B). See, e.g., SEQ ID NOs:32-41. T cells transduced with anti-CD33 DARIC lentiviral vectorsexpress the membrane bound polypeptides shown in FIG. 1A. An anti-CD33scFv CAR or DARIC design was used as a control.

T cells from three donors were each transduced with LVVs encodingdifferent CD33 specific VHH DARICs, an anti-CD33 scFv DARIC, oranti-CD33 scFv CAR and expanded for 10 days. Untransduced T cells, Tcells transduced with anti-CD33 scFv control constructs or anti-CD33 VHHDARIC T cells were stained with recombinant CD33-Fc reagent. Onlycontrol CAR and DARIC T cells were positively stained with the CD33-Fcstaining (FIG. 2A, bottom panel, FIG. 2B). However, most of theanti-CD33 VHH DARIC T cells, but not control CAR or DARIC T cells,stained positively when analyzed with a monoclonal antibody specific forthe VHH domain (FIG. 2A, top panel). Both control CAR and DARIC T cellsand anti-CD33 VHH DARIC T cells had a similar T cell phenotype, asdetermined, in part, by CD62L and CD45RA staining (FIG. 3A and FIG. 3B).

Untransduced T cells, T cells transduced with anti-CD33 scFv controlconstructs or anti-CD33 VHH DARIC T cells were co-cultured with CD33⁺THP-1 cells at an E:T ratio of 1:1 in the presence or absence of AP21967for 24 hours. Anti-CD33 scFv CAR control cells had strong cytokineproduction both in the presence or absence of rapalog. Anti-CD33 scFvDARIC T cells and anti-CD33 VHH DARIC T cells exhibited a robustcytokine response only when cultured with THP-1 cell in the presence ofAP21967 (FIG. 4A and FIG. 4B). Minimal cytokine production was detectedin untransduced controls.

Additionally, the specificity of VHH9 and VHH10 DARICs were assessedagainst full-length CD33 (CD33M) as well as a splice variant thatexpresses a shorter, truncated CD33 (CD33m). Human 293T cells wereelectroporated with mRNA encoding either full length CD33M or the splicevariant CD33m (FIG. 4C). DARIC T cells were co-cultured with themodified 293T cells an E:T ratio of 1:1 in the presence or absence ofAP21967 for 24 hours and assessed for activation as measured by cytokinesecretion (FIG. 4C). The VHH9 DARIC T cells exhibited robust cytokineresponse in response to either CD33M or CD33m 293T cells whereas theVHH10 DARIC T cells were only activated in the presence of CD33M.

Example 2 CD33 VHH DARIC T Cells Specifically Respond to CD33 Antigen

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. Tcells from three donors were each transduced with LVVs encodingdifferent anti-CD33 specific VHH DARICs and expanded for 10 days.Controls included untransduced (UTD) T cells and T cells transduced witha CD33 CAR. The AML cell line MV4-11 normally expresses CD33. MV4-11cells were engineered to knock out the CD33 gene (CD33-KO cells). Theresultant CD33-KO cell line lacked CD33 expression on the cell surface.FIG. 5A. Anti-CD33 VHH DARIC T cells were co-cultured with MV4-11 cellsor CD33-KO cells at an E:T ratio of 1:1 in the presence or absence ofdimerizing drug for 24 hours. Anti-CD33 VHH DARIC T cells producedcytokine in the presence of MV4-11 target cells but not in the presenceof CD33-KO cells. FIG. 5B.

The CD33-KO cell line was modified to express a CD33m splice variant(CD33-KO-C2). MV4-11 cells and CD33-KO-C2 cells were co-cultured withUTD cells, anti-CD33 CAR T cells, or anti-CD33 VHH DARIC T cells in thepresence or absence of dimerizing drug and cytokine production analyzedafter 24 hr. Anti-CD33 VHH9 DARIC recognized both normal CD33 and theCD33m splice variant and produced cytokine when co-cultured with MV4-11cells or CD33-KO-C2 cells (FIG. 5C). Anti-CD33 CAR T cells oranti-CD33VHH2 DARIC control T cells were only active against MV4-11target cells.

Example 3 CD33 VHH DARIC T Cells are not Inhibited by Soluble CD33Protein

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. Tcells from three donors were each transduced with LVVs encodingdifferent anti-CD33 specific VHH DARICs and expanded for 10 days.Anti-CD33 VHH DARIC T cells were co-cultured with CD33⁺ THP-1 cells atan E:T ratio of 1:1 in the presence or absence of rapamycin for 24hours. Various amounts of recombinant CD33-Fc protein were added duringthe co-culture period. Anti-CD33 VHH DARIC T cells exhibited a robustcytokine response in the presence of rapamycin in the presence andabsence of recombinant soluble CD33 protein. FIG. 6.

Example 4 CD33 VHH DARIC T Cells Respond to Low Levels of CD33 Antigen

Anti-CD33 VHH DARIC T cells were generated as described in Example 1. Tcells from three donors were each transduced with LVVs encodingdifferent anti-CD33 specific VHH DARICs and expanded for 10 days.Anti-CD33 VHH DARIC T cells were co-cultured with AP21967 dimerizingagent and CD33^(neg) 293T cells transfected with different amounts ofmRNA encoding CD33. Supernatant was collected after 24 hr and cytokineproduction was analyzed. Anti-CD33 VHH DARIC T cells showeddose-dependent increases in IFNγ production following co-culture withCD33-transfected target cells, even at very low mRNA concentrations(FIG. 7).

Example 5 CD33 VHH DARIC T Cells Control Tumor Growth In Vivo

Anti-CD33 VHH DARIC T cells were generated as described in Example 1.CD33 expressing tumors were established in immunodeficient NSG mice byinoculating the mice with HL60 AML tumor cells expressing a luciferasereporter. Tumor growth was monitored by luminescence. After 10 days,mice were administered 10×10⁶ anti-CD33 VHH DARIC T cell co-administeredwith vehicle or rapamycin. Controls included mice that receivedrapamycin alone or untransduced (UTD) T cells. Tumor growth wascomparable among both treatment and control groups. FIG. 8A. Micetreated with anti-CD33 VHH DARIC T cells and rapamycin showed increasedtumor control compared to mice treated with UTD T cells and rapamycin.FIG. 8B.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A non-natural cell comprising: (a) a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain or a CD4 transmembrane domain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signaling domain; and (b) a second polypeptide comprising: an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide or variant thereof; and a CD4 transmembrane domain or a CD8a transmembrane domain; wherein a bridging factor promotes the formation of a polypeptide complex on the non-natural cell surface with the bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
 2. The non-natural cell of claim 1, wherein the FKBP multimerization domain is FKBP12.
 3. The non-natural cell of claim 1 or claim 2, wherein the FRB polypeptide is FRB T2098L.
 4. The non-natural cell of any one of claims 1 to 3, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
 5. The non-natural cell of any one of claims 1 to 4, wherein the first polypeptide comprises a signal peptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; and a CD3ζ primary signaling domain.
 6. The non-natural cell of any one of claims 1 to 5, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.
 7. The non-natural cell of any one of claims 1 to 6, wherein the second polypeptide comprises a costimulatory domain.
 8. The non-natural cell of claim 7, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRF S25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
 9. The non-natural cell of claim 7 or claim 8, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.
 10. The non-natural cell of any one of claims 1 to 9, wherein the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.
 11. A non-natural cell comprising a polypeptide complex that comprises: (a) a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain or a CD4 transmembrane domain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signaling domain; (b) a second polypeptide comprising: an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide or variant thereof; and a CD4 transmembrane domain or a CD8a transmembrane domain; and (c) a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
 12. The non-natural cell of claim 11, wherein the FKBP multimerization domain is FKBP12.
 13. The non-natural cell of claim 11 or claim 12, wherein the FRB polypeptide is FRB T2098L.
 14. The non-natural cell of any one of claims 11 to 13, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
 15. The non-natural cell of any one of claims 11 to 14, wherein the first polypeptide comprises a signal peptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; and a CD3ζ primary signaling domain.
 16. The non-natural cell of any one of claims 11 to 15, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.
 17. The non-natural cell of any one of claims 11 to 16, wherein the second polypeptide comprises a costimulatory domain.
 18. The non-natural cell of claim 17, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRF S25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
 19. The non-natural cell of claim 17 or claim 18, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.
 20. The non-natural cell of any one of claims 11 to 19, wherein the second polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 22-31.
 21. The non-natural cell of any one of claims 1 to 20, wherein the cell is a hematopoietic cell.
 22. The non-natural cell of any one of claims 1 to 21, wherein the cell is a T cell, an αβ T cell, or a γδ T cell.
 23. The non-natural cell of any one of claims 1 to 22, wherein the cell is a CD3⁺, CD4⁺, and/or CD8⁺ cell.
 24. The non-natural cell of any one of claims 1 to 23, wherein the cell is an immune effector cell.
 25. The non-natural cell of any one of claims 1 to 24, wherein the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cell.
 26. The non-natural cell of any one of claims 1 to 25, wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
 27. The non-natural cell of any one of claims 1 to 26, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
 28. The non-natural cell of any one of claims 1 to 27, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
 29. A fusion polypeptide comprising: (a) a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain or a CD4 transmembrane domain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signaling domain; (b) a polypeptide cleavage signal; and (c) a second polypeptide comprising: an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide or variant thereof; and a CD4 transmembrane domain or a CD8a transmembrane domain.
 30. The fusion polypeptide of claim 29, wherein the FKBP multimerization domain is FKBP12.
 31. The fusion polypeptide of claim 29 or claim 30, wherein the FRB polypeptide is FRB T2098L.
 32. The fusion polypeptide of any one of claims 29 to 31, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
 33. The fusion polypeptide of any one of claims 29 to 32, wherein the first polypeptide comprises a signal peptide, a CD8a transmembrane domain; a CD137 co-stimulatory domain; and a CD3ζ primary signaling domain.
 34. The fusion polypeptide of any one of claims 29 to 33, wherein the second polypeptide comprises a signal peptide and a CD4 transmembrane domain.
 35. The non-natural cell of any one of claims 29 to 34, wherein the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 32-41.
 36. The fusion polypeptide of any one of claims 29 to 35, wherein the second polypeptide comprises a costimulatory domain.
 37. The fusion polypeptide of claim 36, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRF S25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
 38. The fusion polypeptide of claim 36 or claim 37, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.
 39. The fusion polypeptide of any one of claims 29 to 38, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide.
 40. The fusion polypeptide of any one of claims 29 to 39, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.
 41. The fusion polypeptide of any one of claims 29 to 40, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.
 42. The non-natural cell of any one of claims 29 to 41, wherein the fusion polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 42-61.
 43. The fusion polypeptide of any one of claims 29 to 42, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
 44. A polypeptide complex comprising: (a) a first polypeptide comprising: an FRB multimerization domain polypeptide or variant thereof; a CD8a transmembrane domain or a CD4 transmembrane domain; a CD137 co-stimulatory domain; and/or a CD3ζ primary signaling domain; (b) a second polypeptide comprising: an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; an FKBP multimerization domain polypeptide or variant thereof; and a CD4 transmembrane domain or a CD8a transmembrane domain; and (c) a bridging factor associated with and disposed between the multimerization domains of the first and second polypeptides.
 45. The polypeptide complex of claim 44, wherein the FKBP multimerization domain is FKBP12.
 46. The polypeptide complex of claim 44 or claim 45, wherein the FRB polypeptide is FRB T2098L.
 47. The polypeptide complex of any one of claims 44 to 46, wherein the bridging factor is selected from the group consisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.
 48. The polypeptide complex of any one of claims 44 to 47, wherein the first polypeptide comprises a CD8a transmembrane domain; a CD137 co-stimulatory domain; and a CD3ζ primary signaling domain.
 49. The polypeptide complex of any one of claims 44 to 48, wherein the second polypeptide comprises a CD4 transmembrane domain.
 50. The polypeptide complex of any one of claims 44 to 49, wherein the second polypeptide comprises a costimulatory domain.
 51. The polypeptide complex of claim 50, wherein the costimulatory domain of the second polypeptide is selected from a costimulatory molecule selected from the group consisting of: Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRF S25, and zeta chain of T cell receptor associated protein kinase 70 (ZAP70).
 52. The polypeptide complex of claim 50 or claim 51, wherein the costimulatory domain of the second polypeptide is a costimulatory domain isolated from OX40 or TNFR2.
 53. The polypeptide complex of any one of claims 44 to 52, wherein the cell is a hematopoietic cell.
 54. The polypeptide complex of any one of claims 44 to 53, wherein the cell is a T cell, an αβ T cell, or a γδ T cell.
 55. The polypeptide complex of any one of claims 44 to 54, wherein the cell is a CD3⁺, CD4⁺, and/or CD8⁺ cell.
 56. The polypeptide complex of any one of claims 44 to 55, wherein the cell is an immune effector cell.
 57. The polypeptide complex of any one of claims 44 to 56, wherein the cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cell.
 58. The polypeptide complex of any one of claims 44 to 57, wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
 59. The polypeptide complex of any one of claims 44 to 58, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.
 60. The polypeptide complex of any one of claims 44 to 59, wherein the FRB multimerization domain and the FKBP multimerization domain localize extracellularly when of the first polypeptide and the second polypeptide are expressed.
 61. A chimeric antigen receptor (CAR) comprising: a) an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; b) a hinge domain; c) a transmembrane domain; d) one or more intracellular costimulatory signaling domains; and/or e) a primary signaling domain.
 62. The CAR of claim 61, wherein the CAR comprises from 5′ to 3′: a) an anti-CD33 VHH antibody that has an amino acid sequence set forth in any one of SEQ ID NOs: 2-21; b) a hinge domain; c) a transmembrane domain; d) one or more intracellular costimulatory signaling domains; and/or e) a primary signaling domain.
 63. The CAR of claim 61 or claim 62, wherein the hinge domain and transmembrane domain are isolated from CD8a, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD1.
 64. The CAR of any one of claims 61 to 63, wherein the one or more costimulatory signaling domains are isolated from a costimulatory molecule selected from the group consisting of: CD28, CD134, CD137, and CD278.
 65. The CAR of any one of 61 to 64, wherein the CAR comprises a CD8a signal peptide, a CD8a hinge and transmembrane domain, a CD134 costimulatory domain, and a CD3ζ primary signaling domain.
 66. A CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 62-81.
 67. A polynucleotide encoding the first or second polypeptide of any one of claims 1 to 28, the fusion polypeptide of any one of claims 29 to 43, or the CAR of any one of claims 61-66.
 68. A cDNA encoding the first or second polypeptide of any one of claims 1 to 28, the fusion polypeptide of any one of claims 29 to 43, or the CAR of any one of claims 61-66.
 69. An RNA encoding the first or second polypeptide of any one of claims 1 to 28, the fusion polypeptide of any one of claims 29 to 43, or the CAR of any one of claims 61-66.
 70. A vector comprising the polynucleotide of any one of claims 67 to
 69. 71. The vector of claim 70, wherein the vector is an expression vector.
 72. The vector of claim 70, wherein the vector is a transposon.
 73. The vector of claim 72, wherein the vector is a piggyBAC transposon or a Sleeping Beauty transposon.
 74. The vector of claim 70, wherein the vector is a viral vector.
 75. The vector of claim 74, wherein the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes virus vector, a vaccinia virus vector, or a retroviral vector.
 76. The vector of claim 75, wherein the retroviral vector is a lentiviral vector.
 77. The vector of claim 76, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (Hy); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
 78. A cell comprising the first and second polypeptide of any one of claims 1 to 28, the fusion polypeptide of any one of claims 29 to 43, or the CAR of any one of claims 61-66.
 79. The cell of claim 78, wherein the cell is a hematopoietic cell.
 80. The cell of claim 78 or 79, wherein the cell is an immune effector cell.
 81. The cell of any one of claims 78 to 80, wherein the cell is a T cell, an αβ T cell, or a γδ T cell.
 82. The cell of any one of claims 78 to 81, wherein the cell expresses CD3⁺, CD4⁺, CD8⁺, or a combination thereof.
 83. The cell of any one of claims 78 to 82, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
 84. The cell of any one of claims 78 to 83, wherein the cell is a natural killer (NK) cell or natural killer T (NKT) cell.
 85. A composition comprising a cell according to any one of claims 1-28 and 78 to
 84. 86. A composition comprising a physiologically acceptable carrier and a cell according to any one of claims 1-28 and 78 to
 84. 87. A method of treating a subject in need thereof comprising administering the subject an effective amount of the composition of claim 85 or claim
 86. 88. A method of treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith, comprising administering to the subject an effective amount of the composition of claim 85 or claim
 86. 89. A method of treating a solid cancer comprising administering to the subject an effective amount of the composition of claim 85 or claim
 86. 90. The method of claim 89, wherein the solid cancer is selected from the group consisting of: lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer endometrial cancer, or brain cancer.
 91. The method of claim 89 or claim 90, wherein the solid cancer is a non-small cell lung carcinoma, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer endometrial cancer, gliomas, glioblastomas, or oligodendroglioma.
 92. A method of treating a hematological malignancy comprising administering to the subject an effective amount of the composition of claim 85 or claim
 86. 93. The method of claim 92, wherein the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.
 94. The method of claim 92, wherein the hematological malignancy is acute myelogenous leukemia (AML). 